Formulations containing pyridazine compounds

ABSTRACT

The invention relates to chemical compounds, compositions and methods of making and using the same. In particular, the invention provides selected pyridazine compounds of the formula I 
     
       
         
         
             
             
         
       
     
     are independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy, aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo, sulfate, sulfenyl, sulfinyl, sulfonyl, sulfonate, sulfoxide, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S, phosphonate, ureido, carboxyl, carbonyl, carbamoyl, or carboxamide; and X is optionally substituted pyrimidinyl or pyridazinyl, an isomer, a pharmaceutically acceptable salt, or derivative thereof. The invention additional relates to compositions comprising the compounds, and methods of using the compounds and compositions for modulation of cellular pathways, for treatment or prevention of inflammatory diseases, for research, drug screening, and therapeutic applications.

FIELD OF INVENTION

The invention relates to chemical compounds, compositions and methods ofmaking and using the same. In particular, the invention providesselected pyridazine compounds, compositions comprising the compounds,and methods of using the compounds and compositions for modulation ofcellular pathways, for treatment or prevention of inflammatory diseases,for treatment or prevention of neurological conditions, for research,drug screening, and therapeutic applications.

BACKGROUND OF INVENTION

The treatment of neurological conditions and disorders is of greatimportance in medicine and there is a need for new drugs and treatmentsto prevent progression and reverse the impairments of these conditionsand disorders. Neuroinflammation is recognized as a prominent feature inthe pathology of many neurological conditions and diseases.Neuroinflammation is a process that results primarily from abnormallyhigh or chronic activation of glia (microglia and astrocytes). Thisoveractive state of glia results in increased levels of inflammatory andoxidative stress molecules, which can lead to neuron damage or death.Neuronal damage or death can also induce glial activation, facilitatingthe propagation of a localized, detrimental cycle of neuroinflammation(Griffin, W S T et al, Brain Pathol 8: 65-72, 1998). The inflammationcycle has been proposed as a potential therapeutic target in thedevelopment of new approaches to treat inflammatory disease. However,most anti-inflammatory therapeutics developed to date are palliative andprovide minimal, short-lived, symptomatic relief with limited effects oninflammatory disease progression. Thus, there is a need foranti-inflammatory therapeutics that impact disease progression orprevention.

SUMMARY OF INVENTION

The present invention provides certain pyridazine compounds,compositions comprising the compounds, and methods of using thecompounds and compositions for modulation of cellular pathways (e.g.,signal transduction pathways), for treatment or prevention ofinflammatory diseases, for treatment or prevention of neurologicaldiseases and conditions, for research, drug screening, and therapeuticapplications. In particular, the invention generally provides dosageforms, formulations and methods that provide lower risk of side effectsand/or produce beneficial pharmacokinetic profiles, in particular inneuroinflammatory diseases.

The invention contemplates a composition, in particular a formulation ordosage form, effective to provide lower risk of side effects and/or abeneficial pharmacokinetic profile following treatment comprising acompound of the formula I:

wherein R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹², R¹³, and R¹⁴ are independentlyhydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy,aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl,acyloxy, amino, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl,nitro, cyano, halo, sulfate, sulfenyl, sulfinyl, sulfonyl, sulfonate,sulfoxide, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S, phosphonate,ureido, carboxyl, carbonyl, carbamoyl, or carboxamide; and X isoptionally substituted pyrimidinyl or pyridazinyl, an isomer, apharmaceutically acceptable salt, or derivative thereof.

In aspects of the invention, a compound of the formula I is providedwherein R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹², R¹³, and R¹⁴ are independentlyhydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino, imino, azido,thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo, silyl,silyloxy, silylalkyl, silylthio, ═O, ═S, phosphonate, carboxyl,carbonyl, carbamoyl, or carboxamide; and X is pyrimidinyl orpyridazinyl, an isomer, a pharmaceutically acceptable salt, orderivative thereof.

In an aspect, a composition, formulation or dosage form is providedwhich is effective to provide lower risk of side effects and/or abeneficial pharmacokinetic profile following treatment comprising acompound of the formula II:

wherein R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ areindependently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene,alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl,cycloalkoxy, aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, heterocyclic,acyl, acyloxy, amino, imino, azido, thiol, thioalkyl, thioalkoxy,thioaryl, nitro, cyano, halo, sulfenyl, sulfinyl, sulfonyl, sulfonate,sulfate, sulfoxide, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S,phosphonate, ureido, carboxyl, carbonyl, carbamoyl, or carboxamide; oran isomer, a pharmaceutically acceptable salt, or derivative thereof.

In an aspect of the invention, a compound of the formula II is providedwherein R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ areindependently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene,alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy,arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino,imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano,halo, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S, carboxyl,carbonyl, carbamoyl, or carboxamide; or an isomer, a pharmaceuticallyacceptable salt, or derivative thereof.

In aspects of the invention, R¹ in a compound of the formula I or II issubstituted or unsubstituted alkyl, cyclohexyl, aryl, arylalkoxy, aroyl,or heteroaryl.

In a particular aspect, R¹ in a compound of the formula I or II issubstituted or unsubstituted aryl, arylalkoxy, aroyl, or heteroaryl.

In certain aspects of the invention R¹ in a compound of the formula I orII is:

wherein R¹⁵, R¹⁶ and R¹⁷ are independently hydrogen, hydroxyl, alkyl,alkenyl, alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl,cycloalkenyl, cycloalkynyl, cycloalkoxy, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino, imino, azido,thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo, sulfoxide,sulfate, sulfonyl, sulfenyl, sulfinyl, sulfonate, silyl, silyloxy,silylalkyl, silylthio, ═O, ═S, phosphonate, ureido, carboxyl, carbonyl,carbamoyl, or carboxamide.

Therefore, certain aspects of the invention contemplate a composition,in particular a formulation or dosage form, effective to provide lowerrisk of side effects and/or a beneficial pharmacokinetic profilefollowing treatment comprising an amount of a compound of the formulaIII:

wherein R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, andR¹⁷ are independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl,alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl,cycloalkynyl, cycloalkoxy, aryl, aryloxy, arylalkoxy, aroyl, heteroaryl,heterocyclic, acyl, acyloxy, amino, imino, azido, thiol, thioalkyl,thioalkoxy, thioaryl, nitro, cyano, halo, sulfoxide, sulfate, sulfonyl,sulfenyl, sulfinyl, sulfonate, silyl, silyloxy, silylalkyl, silylthio,═O, ═S, ureido, phosphonate, carboxyl, carbonyl, carbamoyl, orcarboxamide.

In general, R⁴R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, andR¹⁷ in a compound of the formula III cannot all be hydrogen.

The invention relates to compounds of the formula I, II or III disclosedherein, in particular pure or substantially pure compounds of theformula I, II or III.

The invention also contemplates utilizing in compositions and methods ofthe invention a compound in FIG. 1, in particular MW01-4-179LKM,MW01-7-084WH, MW01-7-085WH, MW01-7-133WH, MW01-2-151SRM, MW01-5-188WH orMW01-7-057, or isomers, pharmaceutically acceptable salts or derivativesthereof.

A composition of the invention, in particular a formulation or dosageform, may be further characterized by its ability to selectively reduceor block up-regulation of IL-1β and S100B, and/or reduce or prevent lossof PSD-95 and/or synaptophysin.

In aspects, a composition of the invention, in particular a formulationor dosage form, may provide a lower risk of QT-related side effects.

In particular aspects, the invention further provides a composition, inparticular a formulation or dosage form, comprising a compound of theformula I, II or III in a therapeutically effective amount to treat adisease disclosed herein while reducing inhibitory activity at hERGpotassium channel.

In another particular aspect, the invention provides a composition, inparticular a formulation or dosage form, comprising a compound of theformula I, II or III in a therapeutically effective amount to treat adisease disclosed herein while reducing hERG inhibition.

In another particular aspect the invention provides a composition, inparticular a formulation or dosage form, comprising a compound of theformula I, II or III in a therapeutically effective amount to treat adisease disclosed herein in a subject receiving a therapeutic ortreatment that prolongs QT interval.

The invention contemplates a formulation for the treatment of a diseasedisclosed herein comprising a therapeutically effective amount of acompound of the formula I, II or III, to provide a beneficialpharmacokinetic profile, in particular a sustained pharmacokineticprofile, in a pharmaceutically acceptable carrier, excipient, orvehicle. In an aspect, a formulation comprising a compound of theformula I, II or III is provided which is in a form or which has beenadapted for administration to a subject to provide a beneficialpharmacokinetic profile to treat a disease disclosed herein. In anembodiment, a dosage form is provided such that administration of thedosage form to a subject suffering from a disease disclosed hereinprovides a beneficial pharmacokinetic profile resulting in therapeuticeffects including selectively reducing or blocking up-regulation ofIL-1β and S100B, and/or reducing or preventing loss of PSD-95 and/orsynaptophysin over a dosing period. In particular, the composition is ina form adapted to provide a beneficial pharmacokinetic profile thatresults in one or more of the following in a subject for a sustainedtime over a dosing period: selective reduction of up-regulation of IL-1βand S100B, and/or reduction of loss of PSD-95 and/or synaptophysin.

In another aspect, the invention relates to a dosage form comprisingamounts of a compound of the formula I, II or III suitable foradministration to a subject to provide effective concentrations of thecompound in an environment of use or an effective dose that results intherapeutic effects in the prevention, treatment, or control of symptomsof a disease disclosed herein, in particular a neuroinflammatorydisease. In aspects of the invention, the environment of use is thebrain or plasma.

In a further aspect, the invention is directed to a formulation ordosage form suitable for once, twice- or three-times a dayadministration to treat a disease disclosed herein comprising one ormore compound of the formula I, II or III in an amount effective toprovide lower risk of side effects and/or a beneficial pharmacokineticprofile in a dosing period.

In a still further aspect, the invention contemplates a dosage formcomprising one or more compound of the formula I, II or III in an amounteffective to maintain the compound within an effective plasma and/orbrain drug concentration that results in therapeutic effects in thesubject.

The invention additionally relates to a method of preparing a stableformulation or dosage form of a compound of the formula I, II or IIIadapted to provide lower risks of side effects and/or beneficialpharmacokinetic profiles following treatment. Formulations may be placedin an appropriate container and labelled for treatment of an indicateddisease. For administration of a formulation of the invention, suchlabelling would include amount, frequency, and method of administration.

The invention also provides methods to make commercially availableformulations which contain a compound of the formula I, II or III thatprovides lower risk of side effects and/or a beneficial pharmacokineticprofile in the treatment of a disease disclosed herein.

The invention relates to the use of at least one compound of the formulaI, II or III for the preparation of a medicament for providing lowerrisks of side effects and/or a beneficial pharmacokinetic profile intreating a disease disclosed herein. The invention additionally relatesto uses of a pharmaceutical composition of the invention in thepreparation of medicaments for providing lower risks of side effectsand/or a beneficial pharmacokinetic profile in the prevention and/ortreatment of a disease disclosed herein.

Commercially available formulations or medicaments may be pills,tablets, caplets, soft and hard gelatin capsules, lozenges, sachets,cachets, vegicaps, liquid drops, elixirs, suspensions, emulsions,solutions, syrups, aerosols (as a solid or in a liquid medium)suppositories, sterile injectable solutions, and/or sterile packagedpowders, which contain a compound of the formula I, II or III.

Compounds of the formula I, II or III and compositions of the inventionmay be administered therapeutically or prophylactically to treat adisease disclosed herein, in particular neuroinflammatory disease.Therefore the invention provides a method for treating a diseasedisclosed herein, in particular a neuroinflammatory disease, comprisingadministering a therapeutically effective amount or prophylacticallyeffective amount of a compound of the formula I, II or III. In anaspect, the invention provides a method for treating a disease disclosedherein in particular a neuroinflammatory disease comprisingadministering a compound of the formula I, II or III in an amounteffective to lower risks of side effects and/or provide a beneficialpharmacokinetic profile. In an aspect, a method is provided for treatinga disease disclosed herein, in particular a neuroinflammatory disease,comprising administering a compound of the formula I, II or III in anamount effective to selectively inhibit up-regulation of IL-1β andS100B, reduce or prevent loss of PSD-95 and/or synaptophysin, and/orprevent behavioral deficit.

Aspects of the invention provide methods for treating a diseasedisclosed herein, in particular a neuroinflammatory disease, comprisingadministering to a subject a compound of the formula I, II or III in anamount effective to lower risk of QT-related side effects in thesubject. Certain aspects of the invention provide methods for treating adisease disclosed herein, in particular a neuroinflammatory disease,comprising administering to a subject a therapeutically effective amountof a compound of the formula I, II or III to treat the disease whilereducing inhibitory activity at hERG potassium channel. Other aspects ofthe invention provide methods for treating a disease disclosed herein,in particular a neuroinflammatory disease, comprising administering to asubject a therapeutically effective amount of a compound of the formulaI, II or III to treat the disease while reducing hERG inhibition.Further aspects of the invention provide methods for treating a diseasedisclosed herein in a subject suffering from a disease disclosed hereinand receiving a therapeutic or treatment that prolongs QT intervalcomprising administering to the subject a therapeutically effectiveamount of a compound of the formula I to reduce the QT-related sideeffects.

The invention also provides a method for treating and/or preventing adisease disclosed herein in a subject comprising administering to thesubject one or more, in particular two, three or four dosages of aformulation comprising one or more compound of the formula I, II or IIIin an amount effective to maintain the compound within the effectivebrain and/or plasma drug concentration that results in therapeuticeffects in the subject.

In particular aspects of the invention, a method is provided fortreating in a subject a disease involving or characterized byinflammation, in particular neuroinflammation, comprising administeringto the subject a compound of the formula I, II or III in atherapeutically effective amount that provides beneficialpharmacokinetic profiles, in a pharmaceutically acceptable carrier,excipient, or vehicle.

In a further aspect, the invention provides a method involvingadministering to a subject a therapeutic compound of the formula I, IIor III or a pharmaceutically acceptable salt thereof, or a compositioncomprising a compound of the formula I, II or III and a pharmaceuticallyacceptable carrier, excipient, or vehicle which inhibit or reduceneuroflammation, activation of glia, activation of astrocytes,activation of microglia, proimflammatory cytokines, oxidativestress-related enzymes, acute phase proteins and/or components of thecomplement cascade, and provide lower risk of QT-related side effectsand/or a beneficial pharmacokinetic profile.

The invention also provides a kit comprising one or more compound of theformula I, II or III, or a composition of the invention adapted toprovide lower risk of side effects and/or a beneficial pharmacokineticprofile. In an aspect, the invention provides a kit for preventingand/or treating a disorder and/or disease disclosed herein, comprising aformulation or dosage form of the invention, a container, andinstructions for use.

These and other aspects, features, and advantages of the presentinvention should be apparent to those skilled in the art from thefollowing drawings and detailed description.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the structures of MW01-2-151SRM, MW01-6-189WH,MW01-7-107WH, MW01-4-179LKM, MW01-7-084WH, MW01-7-085WH, MW01-7-133WH,and MW01-7-057.

FIG. 2 depicts a synthetic scheme for MW01-3-183WH.

FIG. 3 depicts a synthetic scheme for MW01-2-151SRM.

FIG. 4 depicts a synthetic scheme for MW01-2-151SRM.

FIG. 5 depicts a synthetic scheme for MW01-2-151SRM.

FIG. 6 depicts a synthetic scheme for MW01-2-151SRM.

FIG. 7 depicts a synthetic scheme for MW01-5-188WH.

FIG. 8 depicts a synthetic scheme for MW01-5-188WH.

FIG. 9 depicts a synthetic scheme for MW01-5-188WH.

FIGS. 10A and 10B depict synthetic schemes for MW01-6-189WH

FIG. 11 depicts a synthetic scheme for MW01-7-084WH.

FIG. 12 depicts a synthetic scheme for MW01-7-085WH.

FIG. 13 depicts a synthetic scheme for MW01-7-133WH.

FIG. 14 depicts a synthetic scheme for MW01-7-107WH.

FIG. 15 depicts a synthetic scheme for MW01-7-057.

FIG. 16 show graphs and micrographs illustrating proinflammatorycytokine production by MW01-5-151SRM. (A) Concentration dependentinhibition by MW01-5-151SRM of LPS-induced increases of IL-1β in the BV2microglial cell line. (B) LPS-stimulated accumulation of the NOmetabolite, nitrite, was not inhibited by MW01-5-1151SRM atconcentrations up to 33 μM. (C) MW01-5-1151SRM does not suppressLPS-induced production of iNOS or COX-2 in activated BV-2 cells.

FIG. 17 show graphs and micrographs illustrating proinflammatorycytokine production by MW01-5-189WH. (A) Concentration dependentinhibition by MW01-5-189WH of LPS-induced increases of IL-1β in the BV2microglial cell line. (B) LPS-stimulated accumulation of the NOmetabolite, nitrite, was not inhibited by MW01-5-189WH at concentrationsup to 33 μM. (C) MW01-5-189WH does not suppress LPS-induced productionof iNOS or COX-2 in activated BV-2 cells.

FIG. 18 show graphs and micrographs illustrating proinflammatorycytokine production by MW01-5-107WH. (A) Concentration dependentinhibition by MW01-5-107WH of LPS-induced increases of IL-1β in the BV2microglial cell line. (B) LPS-stimulated accumulation of the NOmetabolite, nitrite, was inhibited by MW01-5-107WH. (C) MW01-5-107WHalso inhibited LPS-induced production of iNOS or COX-2 in activated BV-2cells.

FIG. 19 show graphs and micrographs illustrating proinflammatorycytokine production by MW01-5-179WH. (A) Concentration dependentinhibition by MW01-5-179WH of LPS-induced increases of IL-1β in the BV2microglial cell line. (B) LPS-stimulated accumulation of the NOmetabolite, nitrite, was not inhibited by MW01-5-179WH at concentrationsup to 33 μM. (C) MW01-5-179WH does not suppress LPS-induced productionof iNOS or COX-2 in activated BV-2 cells.

FIG. 20 show graphs and micrographs illustrating proinflammatorycytokine production by MW01-5-084WH. (A) Concentration dependentinhibition by MW01-5-084WH of LPS-induced increases of IL-1β in the BV2microglial cell line. (B) LPS-stimulated accumulation of the NOmetabolite, nitrite, was not inhibited by MW01-5-084WH at concentrationsup to 33 μM. (C) MW01-5-084WH does not suppress LPS-induced productionof iNOS or COX-2 in activated BV-2 cells.

FIG. 21 show graphs and micrographs illustrating proinflammatorycytokine production by MW01-5-085WH. (A) Concentration dependentinhibition by MW01-5-085WH of LPS-induced increases of IL-1β in the BV2microglial cell line. (B) LPS-stimulated accumulation of the NOmetabolite, nitrite, was not inhibited by MW01-5-085WH at concentrationsup to 33 μM. (C) MW01-5-085WH does not suppress LPS-induced productionof iNOS or COX-2 in activated BV-2 cells.

FIG. 22 show graphs and micrographs illustrating proinflammatorycytokine production by MW01-5-0133WH. (A) Concentration dependentinhibition by MW01-5-133WH of LPS-induced increases of IL-1β in the BV2microglial cell line. (B) LPS-stimulated accumulation of the NOmetabolite, nitrite, was not inhibited by MW01-5-133WH at concentrationsup to 33 μM. (C) MW01-5-133WH does not suppress LPS-induced productionof iNOS or COX-2 in activated BV-2 cells.

FIG. 23 show graphs and micrographs illustrating proinflammatorycytokine production by MW01-5-057WH. (A) Concentration dependentinhibition by MW01-5-057WH of LPS-induced increases of IL-1β in the BV2microglial cell line. (B) LPS-stimulated accumulation of the NOmetabolite, nitrite, was not inhibited by MW01-5-057WH at concentrationsup to 33 μM. (C) MW01-5-057WH does not suppress LPS-induced productionof iNOS or COX-2 in activated BV-2 cells.

FIG. 24 A-H shows graphs illustrating in vivo activity of MW01-2-151SRMin the Aβ infusion mouse model. Graphs are of MW01-2-151SRM suppressionof Aβ-induced neuroinflammation and synaptic damage and activity in theY-maze. Hippocampal sections or extracts from vehicle-infused mice(control), Aβ-infused mice injected with solvent, and Aβ-infused miceinjected with MW01-2-151SRM were evaluated for neuroinflammation bymeasurement of the levels of the pro-inflammatory cytokines IL-1β (A),TNFα (B), and S100B (C), and the number of GFAP-positive astrocytes (D),F4/80 (E), the presynaptic marker, synaptophysin (F), and evaluated forsynaptic damage by analysis of the levels of the post-synaptic densityprotein 95 (PSD-95) (G), and Y-maze (H). Data are from one of twoindependent experiments, and are the mean±SEM for 4-5 mice perexperimental group.

FIG. 25 A-E shows graphs illustrating in vivo activity of MW01-2-189SRMin the Aβ infusion mouse model. Graphs are of MW01-2-189SRM suppressionof Aβ-induced neuroinflammation and synaptic damage and activity in theY-maze. Hippocampal sections or extracts from vehicle-infused mice(control), Aβ-infused mice injected with solvent, and Aβ-infused miceinjected with MW01-2-189SRM were evaluated for neuroinflammation bymeasurement of the levels of the pro-inflammatory cytokines IL-1β (A),and S100B (B), the presynaptic marker, synaptophysin (C), and evaluatedfor synaptic damage by analysis of the levels of the post-synapticdensity protein 95 (PSD-95) (D), and Y-maze (E). Data are from threesamples in the MW01-2-189SRM were analyzed.

FIG. 26 A-E shows graphs illustrating in vivo activity of MW01-2-084SRMin the Aβ infusion mouse model. Graphs are of MW01-2-084SRM suppressionof Aβ-induced neuroinflammation and synaptic damage and activity in theY-maze. Hippocampal sections or extracts from vehicle-infused mice(control), Aβ-infused mice injected with solvent, and Aβ-infused miceinjected with MW01-2-084SRM were evaluated for neuroinflammation bymeasurement of the levels of the pro-inflammatory cytokines IL-1β (A),and S100B (B), the presynaptic marker, synaptophysin (C), and evaluatedfor synaptic damage by analysis of the levels of the post-synapticdensity protein 95 (PSD-95) (D), and Y-maze (E). Data are from fivesamples per group analyzed.

FIG. 27 A-E shows graphs illustrating in vivo activity of MW01-2-085SRMin the Aβ infusion mouse model. Graphs are of MW01-2-085SRM suppressionof Aβ-induced neuroinflammation and synaptic damage and activity in theY-maze. Hippocampal sections or extracts from vehicle-infused mice(control), Aβ-infused mice injected with solvent, and Aβ-infused miceinjected with MW01-2-085SRM were evaluated for neuroinflammation bymeasurement of the levels of the pro-inflammatory cytokines IL-1β (A),and S100B (B), the presynaptic marker, synaptophysin (C), and evaluatedfor synaptic damage by analysis of the levels of the post-synapticdensity protein 95 (PSD-95) (D), and Y-maze (E). Data are from threesamples in the MW01-2-085SRM were analyzed.

FIG. 28 A-E shows graphs illustrating in vivo activity of MW01-2-057WHin the Aβ infusion mouse model. Graphs are of MW01-2-057WH suppressionof Aβ-induced neuroinflammation and synaptic damage and activity in theY-maze. Hippocampal sections or extracts from vehicle-infused mice(control), Aβ-infused mice injected with solvent, and Aβ-infused miceinjected with MW01-2-057WH were evaluated for neuroinflammation bymeasurement of the levels of the pro-inflammatory cytokines IL-1β (A),and S100B (B), the presynaptic marker, synaptophysin (C), and evaluatedfor synaptic damage by analysis of the levels of the post-synapticdensity protein 95 (PSD-95) (D), and Y-maze (E). Data are from threesamples in the MW01-2-057SRM were analyzed. There was no significanteffect on PSD-95.

FIG. 29 is a graph showing QTc interval of MW01-2-151SRM (15 mg/10ml/kg/po) (Bazett's). Changes in QTc following oral administration ofMW01-2-151SRM at 15 mg/kg in guinea pigs. QT intervals were correctedfor heart rate changes using Bazett's formula. The broken linesrepresent 95% confidence limits (mean±2SD) for QTc changes in thevehicle (2% Tween 80 in Distilled Water)-treated control. The fivetreated animals are represented by individual symbols.

FIG. 30 is a graph showing QTc interval of Sotalol (0.3 mg/kg/iv)(Bazett's). Changes in QTc following intravenous administration ofSotalol at 0.3 mg/kg in guinea pigs. QT intervals were corrected forheart rate changes using Bazett's formula. The broken lines represent95% confidence limits (mean±2SD) for QTc changes in the vehicle (0.9%NaCl)-treated control. The five treated animals are represented byindividual symbols.

FIG. 31 is a graph showing QTc interval of MW01-2-151SRM (15 mg/10ml/kg/po) (Fredericia's). Changes in QTc following oral administrationof MW01-2-151SRM at 15 mg/kg in guinea pigs. QT intervals were correctedfor heart rate changes using Federicia's formula. The broken linesrepresent 95% confidence limits (mean±2SD) for QTc changes in thevehicle (2% Tween 80 in Distilled Water)-treated control. The fivetreated animals are represented by individual symbols.

FIG. 32 is a graph showing QTc interval of Sotalol (0.3 mg/kg/iv)(Fredericia's). Changes in QTc following intravenous administration ofSotalol at 0.3 mg/kg·in guinea pigs. QT intervals were corrected forheart rate changes using Fredericia's formula. The broken linesrepresent 95% confidence limits (mean±2SD) for QTc changes in thevehicle (0.9% NaCl)-treated control. The five treated animals arerepresented by individual symbols.

FIG. 33 is a graph showing QTc interval of oral administration ofMW01-5-188WH (15 mg/kg p.o.) in guinea pig.

FIG. 34 are graphs of results of liver toxicity studies withMW01-5-188WH, MW01-2-151SRM, and MW01-6-189WH. Compounds wereadministered to C57Bl/6 mice by oral gavage (2.5 mg/kg/day, once dailyfor 2 weeks). Histological liver toxicity was assessed by examination oftissue architecture, cell necrosis, and inflammatory infiltrate. Thescoring scale ranges from 0 (best) to 9 (worst). MW01-5-188WH,MW01-2-151SRM, and MW01-6-189 show no significant differences in livertoxicity score from the control mice receiving either no gavage orvehicle gavage.

FIG. 35 shows that MW01-5-188WH is readily detected in the plasma andthe brain after a single oral dose administration and does not suppressperipheral tissue inflammatory responses or cause liver injury afterchronic oral administration. C57BL/6 mice were administered MW01-5-188WH(2.5 mg/kg) by oral gavage, blood and brain processed at different timesafter administration, and compound levels in plasma and brain determinedas described herein. MW01-5-188WH rapidly appears in plasma (A) andbrain (B), reaches a peak at 15 min, and then slowly declines to basallevels by 120 min. Data are the mean_SEM from three to six mice at eachtime point. MW01-5-188WH does not inhibit increased production of IL-1β(C) and TNF-α (D) in the serum but does suppress the cytokine responsein the brains from the same mice (E, F). Mice (n=3-6 per group) wereadministered by oral gavage either diluent or MW01-5-188WH (2.5 mg/kg)once daily for 2 weeks and then challenged with LPS (10 mg/kg, i.p.) for6 h. Control mice were injected with saline. IL-1β and TNF-α levels inthe serum and in brain supernatants were determined. Data representmean±SEM. ***p_(—)0.001, significantly different from diluent. Dailyoral administration of diluent (G) or MW01-5-188WH(H) (2.5 mg/kg) doesnot result in any histological liver toxicity. Representative liversections from mice treated as in C—F were stained with hematoxylin andeosin. Scale bar, 125 μm. 188, were stained with hematoxylin and eosin.Scale bar, 125 μm. 188, MW01-5-188WH.

FIG. 36 are graphs of stability data using human (A, B) and rat (C, D)microsomes with MW01-2-151SRM in two different amounts, for two timeperiods. E and F show human (E) and (F) rat microsomes withMW01-2-151SRM stability for different time periods compared tominaprine.

FIG. 37 shows a synthetic scheme for synthesis of compounds of theformula I where R¹¹ is benzyl, 4-pyridyl, iso-butyl, or methyl. Reagentsand conditions: a) PhCH₂NH₂NH₂, CH₃COONa, ethanol, reflux, 29 h; b)POCl₃, PCL₅, 120° C., 12 h; c) CH₃COOH, reflux, 5 h; d)1-(2-pyrimidyl)piperazine, 1-butanol, 130° C., 41 h; e) POCl₃, 100° C.,3 h; f) boronic acid, Pd(0). 2 R=benzyl; 3 R=4-pyridyl; 4 R=iso-butyl; 5R=methyl.

FIG. 38 shows a synthetic scheme for synthesis of compounds of theformula I where R¹ is methyl.

FIG. 39 shows a synthetic scheme for synthesis of pyrazine analogs ofthe invention. a) NaOH, −41° C., MeOH; b) Tf₂O, DMAP, Pyridine, rt; c)1-(2-pyrimidyl)piperazine, DMSO, 60° C.

DETAILED DESCRIPTION OF EMBODIMENTS

For convenience, certain terms employed in the specification, examples,and appended claims are collected here.

Numerical ranges recited herein by endpoints include all numbers andfractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbersand fractions thereof are presumed to be modified by the term “about.”The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%,preferably 10-20%, more preferably 10% or 15%, of the number to whichreference is being made. Further, it is to be understood that “a,” “an,”and “the” include plural referents unless the content clearly dictatesotherwise. Thus, for example, reference to a composition comprising “acompound” includes a mixture of two or more compounds.

As used herein the terms “administering” and “administration” refer to aprocess by which a therapeutically effective amount of a compound of theformula I, II or III or composition contemplated herein is delivered toa subject for prevention and/or treatment purposes. Compositions areadministered in accordance with good medical practices taking intoaccount the subject's clinical condition, the site and method ofadministration, dosage, patient age, sex, body weight, and other factorsknown to physicians.

As used herein, the term “co-administration” of “co-administered” refersto the administration of at least two compounds or agent(s) or therapiesto a subject. In some embodiments, the co-administration of two or moreagents/therapies is concurrent. In other embodiments, a firstagent/therapy is administered prior to a second agent/therapy. In thisaspect, each component may be administered separately, but sufficientlyclose in time to provide the desired effect, in particular a beneficial,additive, or synergistic effect. Those of skill in the art understandthat the formulations and/or routes of administration of the variousagents/therapies used may vary. The appropriate dosage forco-administration can be readily determined by one skilled in the art.In some embodiments, when agents/therapies are co-administered, therespective agents/therapies are administered at lower dosages thanappropriate for their administration alone. Thus, co-administration isespecially desirable in embodiments where the co-administration of theagents/therapies lowers the requisite dosage of a known potentiallyharmful (e.g., toxic) agent(s).

The term “treating” refers to reversing, alleviating, or inhibiting theprogress of a disease, or one or more symptoms of such disease, to whichsuch term applies. Depending on the condition of the subject, the termalso refers to preventing a disease, and includes preventing the onsetof a disease, or preventing the symptoms associated with a disease. Atreatment may be either performed in an acute or chronic way. The termalso refers to reducing the severity of a disease or symptoms associatedwith such disease prior to affliction with the disease. Such preventionor reduction of the severity of a disease prior to affliction refers toadministration of a compound or composition of the present invention toa subject that is not at the time of administration afflicted with thedisease. “Preventing” also refers to preventing the recurrence of adisease or of one or more symptoms associated with such disease.“Treatment” and “therapeutically,” refer to the act of treating, as“treating” is defined above. The purpose of prevention and interventionis to combat the disease, condition, or disorder and includes theadministration of an active compound to prevent or delay the onset ofthe symptoms or complications, or alleviating the symptoms orcomplications, or eliminating the disease, condition, or disorder.

The terms “subject”, “individual”, or “patient” are used interchangeablyherein and refer to an animal preferably a warm-blooded animal such as amammal. Mammal includes without limitation any members of the Mammalia.A mammal, as a subject or patient in the present disclosure, can be fromthe family of Primates, Carnivora, Proboscidea, Perissodactyla,Artiodactyla, Rodentia, and Lagomorpha. Among other specific embodimentsa mammal of the present invention can be Canis familiaris (dog), Feliscatus (cat), Elephas maximus (elephant), Equus caballus (horse), Susdomesticus (pig), Camelus dromedarious (camel), Cervus axis (deer),Giraffa camelopardalis (giraffe), Bos taurus (cattle/cows), Capra hircus(goat), Ovis aries (sheep), Mus musculus (mouse), Lepus brachyurus(rabbit), Mesocricetus auratus (hamster), Cavia porcellus (guinea pig),Meriones unguiculatus (gerbil), or Homo sapiens (human). In a particularembodiment, the mammal is a human. In other embodiments, animals can betreated; the animals can be vertebrates, including both birds andmammals. In aspects of the invention, the terms include domestic animalsbred for food or as pets, including equines, bovines, sheep, poultry,fish, porcines, canines, felines, and zoo animals, goats, apes (e.g.gorilla or chimpanzee), and rodents such as rats and mice.

Typical subjects for treatment include persons afflicted with orsuspected of having or being pre-disposed to a disease disclosed herein,or persons susceptible to, suffering from or that have suffered adisease disclosed herein. A subject may or may not have a geneticpredisposition for a disease disclosed herein. In the context of certainaspects of the invention, the term “subject” generally refers to anindividual who will receive or who has received treatment (e.g.,administration of a compound of the formula I, II or III, and optionallyone or more other agents) for a condition characterized by inflammation,the dysregulation of protein kinase activity, and/or dysregulation ofapototic processes. In certain aspects, a subject may be a healthysubject.

In particular aspects, a subject shows signs of cognitive deficits andAlzheimer's disease neuropathology. In embodiments of the invention thesubjects are suspectible to, or suffer from Alzheimer's disease.

As utilized herein, the term “healthy subject” means a subject, inparticular a mammal, having no diagnosed disease, disorder, infirmity,or ailment, more particularly a disease, disorder, infirmity or ailmentknown to impair or otherwise diminish memory.

The term “diagnosed,” as used herein, refers to the recognition of adisease by its signs and symptoms (e.g., resistance to conventionaltherapies), or genetic analysis, pathological analysis, histologicalanalysis, and the like.

As used herein, the term “modulate” refers to the activity of a compound(e.g., a compound of the formula I, II or III) to affect (e.g., topromote or retard) an aspect of cellular function, including, but notlimited to, cell growth, proliferation, apoptosis, and the like.

“Therapeutically effective amount” relates to the amount or dose of anactive compound of the formula I, II or III or composition comprisingthe same, that will lead to one or more desired effects, in particular,one or more therapeutic effects or beneficial pharmacokinetic profiles.A therapeutically effective amount of a substance can vary according tofactors such as the disease state, age, sex, and weight of the subject,and the ability of the substance to elicit a desired response in thesubject. A dosage regimen may be adjusted to provide the optimumtherapeutic response or pharmacokinetic profile. For example, severaldivided doses may be administered daily or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

The term “prophylactically effective amount” refers to an amounteffective, at dosages and for periods of time necessary, to achieve thedesired prophylactic result. Typically, since a prophylactic dose isused in subjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

The term “beneficial pharmacokinetic profile” refers to amounts or dosesof a compound of the formula I, II or III that provide levels of thecompound in plasma and/or brain or a required dose resulting intherapeutic effects in the prevention, treatment, or control of symptomsof a disease disclosed herein, in particular a neuroinflammatorydisease, more particularly Alzheimer's disease. The term “sustainedpharmacokinetic profile” as used herein refers to a length of timeefficacious levels of a biologically active compound of the formula I,II or III is in its environment of use. A sustained pharmacokineticprofile can be such that a single or twice daily administrationadequately prevents, treats, or controls symptoms of a disease disclosedherein. A beneficial pharmacokinetic profile may provide therapeuticallyeffective amounts of the compound of the formula I, II or III in theplasma and/or brain for about 12 to about 48 hours, 12 hours to about 36hours, or 12 hours to about 24 hours.

A “therapeutic effect” refers to an effect of a composition, inparticular a formulation or dosage form, or method disclosed herein,including improved biological activity, efficacy, and/or lower risk ofside effects (e.g., lower risk of QT-related side effects). Atherapeutic effect may be a sustained therapeutic effect that correlateswith a continuous plasma and/or brain concentration of a compound of theformula I, II or III over a dosing period, in particular a sustaineddosing period. A therapeutic effect may be a statistically significanteffect in terms of statistical analysis of an effect of a compound ofthe formula I, II or III versus the effects without the compound.

“Statistically significant” or “significantly different” effects orlevels may represent levels that are higher or lower than a standard. Inaspects of the invention, the difference may be 1.5, 2, 3, 4, 5, 6, 7,8, 9, 10, 15, 20, 25 or 50 times higher or lower compared with theeffect obtained without a compound of the formula I, II or III.

In an embodiment, where the disease is neuroinflammatory disease such asAlzheimer's disease, therapeutic effects of a compound or composition ortreatment of the invention can manifest as one, two, three, four, five,six, seven, eight, or all of the following, in particular five or more,more particularly seven or more of the following:

-   -   a) A reduction in protein kinase activity (e.g. DAPK), in        particular at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%,        15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%,        or 99% decrease in protein kinase activity.    -   b) A reduction in glial activation response, in particular, at        least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%,        33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%        reduction in glial activation response.    -   c) A reduction in glial activity in the brain, relative to the        levels determined in the absence of a compound of the formula I,        II or III in subjects with symptoms of a neuroinflammatory        disease. In particular, the compounds induce at least about a        2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%        decrease in glial activity.    -   d) A reduction in astrocyte activation response, in particular,        at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%,        30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%        reduction in astrocyte activation response.    -   e) A reduction in astrocyte activity in the brain, relative to        the levels determined in the absence of a compound or treatment        according to the invention. In particular, the compounds induce        at least about a 2%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%,        80%, or 90% decrease in astrocyte activity.    -   f) A reduction in microglial activation, in particular, at least        about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%,        35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% reduction in        microglial activation.    -   g) A reduction in microglial activation response, in particular,        at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%,        30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%        reduction in microglial activation response.    -   h) A reduction in loss of synaptophysin and/or PSD-95, in        particular at least about a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%,        15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%,        or 99% reduction in loss of synaptophysin and/or PSD-95.    -   i) A reduction in oxidative stress-related responses (e.g.,        nitric oxide synthase production and/or nitric oxide        accumulation), in particular at least about a 0.05%, 0.1%, 0.5%,        1%, 2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%,        70%, 80%, 90%, 95%, or 99% reduction in oxidative stress-related        responses such as nitric oxide synthase production and nitric        oxide accumulation.    -   j) A reduction in cellular apoptosis and/or death associated        protein kinase activity, in particular a 0.05%, 0.1%, 0.5%, 1%,        2%, 5%, 10%, 15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%,        80%, 90%, 95%, or 99% reduction in cellular apoptosis and/or        death associated protein kinase activity.    -   k) A reduction in proinflammatory cytokine responses in        particular a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 30%,        33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%, or 99%        reduction in proinflammatory cytokine responses.    -   l) A reduction in interleukin-1β and/or tumor necrosis factor a        production in particular a 0.05%, 0.1%, 0.5%, 1%, 2%, 5%, 10%,        15%, 20%, 30%, 33%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 95%,        or 99% reduction in interleukin-1β and/or tumor necrosis factor        α production.    -   m) A slowing of the rate of disease progression in a subject        with a neuroinflammatory disease (e.g., Alzheimer's disease).    -   n) Increase in survival in a subject with symptoms of a        neuroinflammatory disease (e.g., Alzheimer's disease).

In particular aspects of the invention therapeutic effects of compounds,compositions or treatments of the invention can manifest as (a) and (b);(a), (b) and (c); (a) through (d); (a) through (e); (a) through (f); (a)through (g); (a) through (h); (a) through (i), (a) through (j), and (a)through (k), (a) through (l), (a) through (m), or (a) through (n).

The term “pharmaceutically acceptable carrier, excipient, or vehicle”refers to a medium which does not interfere with the effectiveness oractivity of an active ingredient and which is not toxic to the hosts towhich it is administered. A carrier, excipient, or vehicle includesdiluents, binders, adhesives, lubricants, disintegrates, bulking agents,wetting or emulsifying agents, pH buffering agents, and miscellaneousmaterials such as absorbants that may be needed in order to prepare aparticular composition. Examples of carriers etc. include but are notlimited to saline, buffered saline, dextrose, water, glycerol, ethanol,and combinations thereof. The use of such media and agents for an activesubstance is well known in the art.

The compounds of the formula I, II or III disclosed herein also include“pharmaceutically acceptable salt(s)”. By pharmaceutically acceptablesalts is meant those salts which are suitable for use in contact withthe tissues of a subject or patient without undue toxicity, irritation,allergic response and the like, and are commensurate with a reasonablebenefit/risk ratio. Pharmaceutically acceptable salts are described forexample, in S. M. Berge, et al., J. Pharmaceutical Sciences, 1977, 66:1.Suitable salts include salts that may be formed where acidic protons inthe compounds are capable of reacting with inorganic or organic bases.Suitable inorganic salts include those formed with alkali metals, e.g.sodium and potassium, magnesium, calcium, and aluminum. Suitable organicsalts include those formed with organic bases such as the amine bases,e.g. ethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like. Suitable salts also include acidaddition salts formed with inorganic acids (e.g. hydrochloric andhydrobromic acids) and organic acids (e.g. acetic acid, citric acid,maleic acid, and the alkane- and arene-sulfonic acids such asmethanesulfonic acid and benezenesulfonic acid). When there are twoacidic groups present, a pharmaceutically acceptable salt may be amono-acid-mono-salt or a di-salt; and similarly where there are morethan two acidic groups present, some or all of such groups can besalified.

A compound of the formula I, II or III can contain one or moreasymmetric centers and may give rise to enantiomers, diasteriomers, andother stereoisomeric forms which may be defined in terms of absolutestereochemistry as (R)- or (S)-. Thus, compounds of the formula I, II orIII include all possible diasteriomers and enantiomers as well as theirracemic and optically pure forms. Optically active (R)- and (S)-isomersmay be prepared using chiral synthons or chiral reagents, or resolvedusing conventional techniques. When a compound of the formula I, II orIII contains centers of geometric asymmetry, and unless specifiedotherwise, it is intended that the compounds include both E and Ageometric isomers. All tautomeric forms are also included within thescope of a compound of the formula I, II or III.

A compound of the formula I, II or III includes crystalline forms whichmay exist as polymorphs. Solvates of the compounds formed with water orcommon organic solvents are also intended to be encompassed within theterm. In addition, hydrate forms of the compounds and their salts areencompassed within this invention. Further prodrugs of compounds of theformula I, II or III are encompassed within the term.

The term “solvate” means a physical association of a compound with oneor more solvent molecules or a complex of variable stoichiometry formedby a solute (for example, a compound of the invention) and a solvent,for example, water, ethanol, or acetic acid. This physical associationmay involve varying degrees of ionic and covalent bonding, includinghydrogen bonding. In certain instances, the solvate will be capable ofisolation, for example, when one or more solvent molecules areincorporated in the crystal lattice of the crystalline solid. Ingeneral, the solvents selected do not interfere with the biologicalactivity of the solute. Solvates encompass both solution-phase andisolatable solvates. Representative solvates include hydrates,ethanolates, methanolates, and the like. Dehydrate, co-crystals,anhydrous, or amorphous forms of the compounds of the invention are alsoincluded. The term “hydrate” means a solvate wherein the solventmolecule(s) is/are H₂O, including, mono-, di-, and various poly-hydratesthereof. Solvates can be formed using various methods known in the art.

Crystalline compounds of the formula I, II or III can be in the form ofa free base, a salt, or a co-crystal. Free base compounds can becrystallized in the presence of an appropriate solvent in order to forma solvate. Acid salt compounds of the formula I, II or III (e.g. HCl,HBr, benzoic acid) can also be used in the preparation of solvates. Forexample, solvates can be formed by the use of acetic acid or ethylacetate. The solvate molecules can form crystal structures via hydrogenbonding, van der Waals forces, or dispersion forces, or a combination ofany two or all three forces.

The amount of solvent used to make solvates can be determined by routinetesting. For example, a monohydrate of a compound of the formula I, IIor III would have about 1 equivalent of solvent (H₂O) for eachequivalent of a compound of the invention. However, more or less solventmay be used depending on the choice of solvate desired.

Compounds of the formula I, II or III may be amorphous or may havedifferent crystalline polymorphs, possibly existing in differentsolvation or hydration states. By varying the form of a drug, it ispossible to vary the physical properties thereof. For example,crystalline polymorphs typically have different solubilities from oneanother, such that a more thermodynamically stable polymorph is lesssoluble than a less thermodynamically stable polymorph. Pharmaceuticalpolymorphs can also differ in properties such as shelf-life,bioavailability, morphology, vapor pressure, density, color, andcompressibility.

The term “prodrug” means a covalently-bonded derivative or carrier ofthe parent compound or active drug substance which undergoes at leastsome biotransformation prior to exhibiting its pharmacologicaleffect(s). In general, such prodrugs have metabolically cleavable groupsand are rapidly transformed in vivo to yield the parent compound, forexample, by hydrolysis in blood, and generally include esters and amideanalogs of the parent compounds. The prodrug is formulated with theobjectives of improved chemical stability, improved patient acceptanceand compliance, improved bioavailability, prolonged duration of action,improved organ selectivity, improved formulation (e.g., increasedhydrosolubility), and/or decreased side effects (e.g., toxicity). Ingeneral, prodrugs themselves have weak or no biological activity and arestable under ordinary conditions. Prodrugs can be readily prepared fromthe parent compounds using methods known in the art, such as thosedescribed in A Textbook of Drug Design and Development,Krogsgaard-Larsen and H. Bundgaard (eds.), Gordon & Breach, 1991,particularly Chapter 5: “Design and Applications of Prodrugs”; Design ofProdrugs, H. Bundgaard (ed.), Elsevier, 1985; Prodrugs: Topical andOcular Drug Delivery, K. B. Sloan (ed.), Marcel Dekker, 1998; Methods inEnzymology, K. Widder et al. (eds.), Vol. 42, Academic Press, 1985,particularly pp. 309 396; Burger's Medicinal Chemistry and DrugDiscovery, 5th Ed., M. Wolff (ed.), John Wiley & Sons, 1995,particularly Vol. I and pp. 172 178 and pp. 949 982; Pro-Drugs as NovelDelivery Systems, T. Higuchi and V. Stella (eds.), Am. Chem. Soc., 1975;and Bioreversible Carriers in Drug Design, E. B. Roche (ed.), Elsevier,1987.

Examples of prodrugs include, but are not limited to esters (e.g.,acetate, formate, and benzoate derivatives), carbamates (e.g.N,N-dimethylaminocarbonyl) of hydroxy functional groups on compounds ofthe formula I, II or III, and the like

A compound of the formula I, II or III can include a pharmaceuticallyacceptable co-crystal or a co-crystal salt. A pharmaceuticallyacceptable co-crystal includes a co-crystal that is suitable for use incontact with the tissues of a subject or patient without undue toxicity,irritation, allergic response and has the desired pharmacokineticproperties.

The term “co-crystal” as used herein means a crystalline materialcomprised of two or more unique solids at room temperature, eachcontaining distinctive physical characteristics, such as structure,melting point, and heats of fusion. Co-crystals can be formed by anactive pharmaceutical ingredient (API) and a co-crystal former either byhydrogen bonding or other non-covalent interactions, such as pi stackingand van der Waals interactions. An aspect of the invention provides fora co-crystal wherein the co-crystal former is a second API. In anotheraspect, the co-crystal former is not an API. In another aspect, theco-crystal comprises more than one co-crystal former. For example, two,three, four, five, or more co-crystal formers can be incorporated in aco-crystal with an API. Pharmaceutically acceptable co-crystals aredescribed, for example, in “Pharmaceutical co-crystals,” Journal ofPharmaceutical Sciences, Volume 95 (3) Pages 499-516, 2006. The methodsproducing co-crystals are discussed in the United States PatentApplication 20070026078.

A co-crystal former which is generally a pharmaceutically acceptablecompound, may be, for example, benzoquinone, terephthalaldehyde,saccharin, nicotinamide, acetic acid, formic acid, butyric acid,trimesic acid, 5-nitroisophthalic acid,adamantane-1,3,5,7-tetracarboxylic acid, formamide, succinic acid,fumaric acid, tartaric acid, malic acid, tartaric acid, malonic acid,benzamide, mandelic acid, glycolic acid, fumaric acid, maleic acid,urea, nicotinic acid, piperazine, p-phthalaldehyde,2,6-pyridinecarboxylic acid, 5-nitroisophthalic acid, citric acid, andthe alkane- and arene-sulfonic acids such as methanesulfonic acid andbenezenesulfonic acid.

In general, all physical forms of compounds of the formula I, II or IIIare intended to be within the scope of the present invention.

A compound of the formula I, II or III may be pure or substantiallypure. As used herein, the term “pure” in general means better than 90%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% pure, and “substantially pure”means a compound synthesized such that the compound, as made or asavailable for consideration into a composition or therapeutic dosagedescribed herein, has only those impurities that can not readily norreasonably be removed by conventional purification processes.

“Optional” or “optionally” means that the subsequently described eventor circumstance may but need not occur, and that the descriptionincludes instances where the event or circumstance occurs and instancesin which it does not occur. For example, “alkyl group optionallysubstituted with a halo group” means that the halo may but need not bepresent, and the description includes situations where the alkyl groupis substituted with a halo group and situations where the alkyl group isnot substituted with the halo group.

A compound of the formula I, II or III includes derivatives. As usedherein the term “derivative” of a compound of the formula I, II or IIIrefers to a chemically modified compound wherein the chemicalmodification takes place either at a functional group or ring of thecompound. Non-limiting examples of derivatives of compounds of theformula I, II or III may include N-acetyl, N-methyl, N-hydroxy groups atany of the available nitrogens in the compound. Derivative groups thatmay be used to modify the compounds of the formula I, II or III can befound in U.S. Patent Application No. 20030176437 (herein incorporated byreference in its entirety for all purposes).

In aspects of the invention, a compound of the formula I, II or III is apharmaceutically functional derivative. A “pharmaceutically functionalderivative” includes any pharmaceutically acceptable derivative of acompound of the formula I, II or III, for example, an ester or an amide,which upon administration to a subject is capable of providing (directlyor indirectly) a compound of the formula I, II or III or an activemetabolite or residue thereof. Such derivatives are recognizable tothose skilled in the art, without undue experimentation (see for exampleBurger's Medicinal Chemistry and Drug Discovery, 5.sup.th Edition, Vol1: Principles and Practice, which has illustrative pharmaceuticallyfunctional derivatives).

A compound of the formula I, II or III may include a carrier. Suitablecarriers include a polymer, carbohydrate, or a peptide.

A “polymer” refers to molecules comprising two or more monomer subunitsthat may be identical repeating subunits or different repeatingsubunits. A monomer generally comprises a simple structure,low-molecular weight molecule containing carbon. Polymers may optionallybe substituted. Polymers that can be used in the present inventioninclude without limitation vinyl, acryl, styrene, carbohydrate derivedpolymers, polyethylene glycol (PEG), polyoxyethylene, polymethyleneglycol, poly-trimethylene glycols, polyvinylpyrrolidone,polyoxyethylene-polyoxypropylene block polymers, and copolymers, salts,and derivatives thereof. In aspects of the invention, the polymer ispoly(2-acrylamido-2-methyl-1-propanesulfonic acid);poly(2-acrylamido-2-methyl-1-propanesulfonic acid-coacrylonitrile,poly(2-acrylamido-2-methyl-1-propane sulfonic acid-co-styrene),poly(vinylsulfonic acid); poly(sodium 4-styrenesulfonic acid); andsulfates and sulfonates derived therefrom; poly(acrylic acid),poly(methylacrylate), poly(methyl methacrylate), and poly(vinylalcohol).

A “carbohydrate” as used herein refers to a polyhydroxyaldehyde, orpolyhydroxyketone and derivatives thereof. The term includesmonosaccharides such as erythrose, arabinose, allose, altrose, glucose,mannose, threose, xylose, gulose, idose, galactose, talose, aldohexose,fructose, ketohexose, ribose, and aldopentose. The term also includescarbohydrates composed of monosaccharide units, including disaccharides,oligosaccharides, or polysaccharides. Examples of disaccharides aresucrose, lactose, and maltose. Oligosaccharides generally containbetween 3 and 9 monosaccharide units and polysaccharides contain greaterthan 10 monosaccharide units. A carbohydrate group may be substituted atone two, three or four positions, other than the position of linkage toa compound of the formula I, II or III. For example, a carbohydrate maybe substituted with one or more alkyl, amino, nitro, halo, thiol,carboxyl, or hydroxyl groups, which are optionally substituted.Illustrative substituted carbohydrates are glucosamine, orgalactosamine. In aspects of the invention, the carbohydrate is a sugar,in particular a hexose or pentose and may be an aldose or a ketose. Asugar may be a member of the D or L series and can include amino sugars,deoxy sugars, and their uronic acid derivatives. In embodiments of theinvention where the carbohydrate is a hexose, the hexose is glucose,galactose, or mannose, or substituted hexose sugar residues such as anamino sugar residue such as hexosamine, galactosamine, glucosamine, inparticular D-glucosamine (2-amino-2-doexy-D-glucose) or D-galactosamine(2-amino-2-deoxy-D-galactose). Illustrative pentose sugars includearabinose, fucose, and ribose.

A sugar residue may be linked to a compound of the formula I, II or IIIfrom a 1,1 linkage, 1,2 linkage, 1,3 linkage, 1,4 linkage, 1,5 linkage,or 1,6 linkage. A linkage may be via an oxygen atom of a compound of theformula I, II or III. An oxygen atom can be replaced one or more timesby —CH₂— or —S— groups.

The term “carbohydrate” also includes glycoproteins such as lectins(e.g. concanavalin A, wheat germ agglutinin, peanutagglutinin,seromucoid, and orosomucoid) and glycolipids such as cerebroside andganglioside.

A “peptide” carrier for use in the practice of the present inventionincludes one, two, three, four, or five or more amino acids covalentlylinked through a peptide bond. A peptide can comprise one or morenaturally occurring amino acids, and analogs, derivatives, and congenersthereof. A peptide can be modified to increase its stability,bioavailability, solubility, etc. “Peptide analogue” and “peptidederivative” as used herein include molecules which mimic the chemicalstructure of a peptide and retain the functional properties of thepeptide. A carrier for use in the present invention can be an amino acidsuch as alanine, glycine, proline, methionine, serine, threonine,histidine, asparagine, alanyl-alanyl, prolyl-methionyl, orglycyl-glycyl. A carrier can be a polypeptide such as albumin,antitrypsin, macroglobulin, haptoglobin, caeruloplasm, transferring, α-or β-lipoprotein, β- or γ-globulin or fibrinogen.

Approaches to designing peptide analogues, derivatives and mimetics areknown in the art. For example, see Farmer, P. S. in Drug Design (E. J.Ariens, ed.) Academic Press, New York, 1980, vol. 10, pp. 119-143; Ball.J. B. and Alewood, P. F. (1990) J. Mol. Recognition. 3:55; Morgan, B. A.and Gainor, J. A. (1989) Ann. Rep. Med. Chem. 24:243; and Freidinger, R.M. (1989) Trends Pharmacol. Sci. 10:270. See also Sawyer, T. K. (1995)“Peptidomimetic Design and Chemical Approaches to Peptide Metabolism” inTaylor, M. D. and Amidon, G. L. (eds.) Peptide-Based Drug Design:Controlling Transport and Metabolism, Chapter 17; Smith, A. B. 3rd, etal. (1995) J. Am. Chem. Soc. 117:11113-11123; Smith, A. B. 3rd, et al.(1994) J. Am. Chem. Soc. 116:9947-9962; and Hirschman, R., et al. (1993)J. Am. Chem. Soc. 115:12550-12568.

A peptide can be attached to a compound of the formula I, II or IIIthrough a functional group on the side chain of certain amino acids(e.g. serine) or other suitable functional groups. A carrier maycomprise four or more amino acids with groups attached to three or moreof the amino acids through functional groups on side chains. In anaspect, the carrier is one amino acid, in particular a sulfonatederivative of an amino acid, for example cysteic acid.

The term “alkyl”, either alone or within other terms such as “thioalkyl”and “arylalkyl”, means a monovalent, saturated hydrocarbon radical whichmay be a straight chain (i.e. linear) or a branched chain. An alkylradical for use in the present invention generally comprises from about1 to 20 carbon atoms, particularly from about 1 to 10, 1 to 8 or 1 to 7,more particularly about 1 to 6 carbon-atoms, or 3 to 6. Illustrativealkyl radicals include methyl, ethyl, n-propyl, n-butyl, n-pentyl,n-hexyl, isopropyl, isobutyl, isopentyl, amyl, sec-butyl, tert-butyl,tert-pentyl, n-heptyl, n-octyl, n-nonyl, n-decyl, undecyl, n-dodecyl,n-tetradecyl, pentadecyl, n-hexadecyl, heptadecyl, n-octadecyl,nonadecyl, eicosyl, dosyl, n-tetracosyl, and the like, along withbranched variations thereof. In certain aspects of the invention analkyl radical is a C₁-C₆ lower alkyl comprising or selected from thegroup consisting of methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl,isopropyl, isobutyl, isopentyl, amyl, tributyl, sec-butyl, tert-butyl,tert-pentyl, and n-hexyl. An alkyl radical may be optionally substitutedwith substituents as defined herein at positions that do notsignificantly interfere with the preparation of compounds of the formulaI, II or III and do not significantly reduce the efficacy of thecompounds. In certain aspects of the invention, an alkyl radical issubstituted with one to five substituents including halo, lower alkoxy,lower aliphatic, a substituted lower aliphatic, hydroxy, cyano, nitro,thio, amino, keto, aldehyde, ester, amide, substituted amino, carboxyl,sulfonyl, sulfinyl, sulfenyl, sulfate, sulfoxide, substituted carboxyl,halogenated lower alkyl (e.g. CF₃), halogenated lower alkoxy,hydroxycarbonyl, lower alkoxycarbonyl, lower alkylcarbonyloxy, loweralkylcarbonylamino, cycloaliphatic, substituted cycloaliphatic, or aryl(e.g., phenylmethyl (i.e. benzyl)), heteroaryl (e.g., pyridyl), andheterocyclic (e.g., piperidinyl, morpholinyl). Substituents on an alkylgroup may themselves be substituted.

In aspects of the invention, “substituted alkyl” includes an alkyl groupsubstituted by, for example, one to five substituents, and preferably 1to 3 substituents, such as alkyl, alkoxy, oxo, alkanoyl, aryl, aralkyl,aryloxy, alkanoyloxy, cycloalkyl, acyl, amino, hydroxyamino, alkylamino,arylamino, alkoxyamino, aralkylamino, cyano, halogen, hydroxyl,carboxyl, carbamyl, carboxylalkyl, keto, thioketo, thiol, alkylthiol,arylthio, aralkylthio, sulfonamide, thioalkoxy, and nitro.

In respect to certain aspects of the invention, the term “substitutedaliphatic” refers to an alkyl or an alkane possessing less than 10carbons. The term “substituted aliphatic” refers to an alkyl or analkane possessing less than 10 carbons where at least one of thealiphatic hydrogen atoms has been replaced by a halogen, an amino, ahydroxy, a nitro, a thio, a ketone, an aldehyde, an ester, an amide, alower aliphatic, a substituted lower aliphatic, or a ring (aryl,substituted aryl, cycloaliphatic, or substituted cycloaliphatic, etc.).Examples of such groups include, but are not limited to, 1-chloroethyland the like.

As used herein in respect to certain aspects of the invention, the term“lower-alkyl-substituted-amino” refers to any alkyl unit containing upto and including eight carbon atoms where one of the aliphatic hydrogenatoms is replaced by an amino group. Examples of such include, but arenot limited to, ethylamino and the like.

As used herein in respect to certain aspects of the invention, the term“lower-alkyl-substituted-halogen” refers to any alkyl chain containingup to and including eight carbon atoms where one of the aliphatichydrogen atoms is replaced by a halogen. Examples of such include, butare not limited to, chlorethyl and the like.

As used herein, the term “acetylamino” shall mean any primary orsecondary amino that is acetylated. Examples of such include, but arenot limited to, acetamide and the like.

As used herein the term “alkenyl” refers to an unsaturated, acyclicbranched or straight-chain hydrocarbon radical comprising at least onedouble bond. An alkenyl radical may contain from about 2 to 24 or 2 to10 carbon atoms, in particular from about 3 to 8 carbon atoms and moreparticularly about 3 to 6 or 2 to 6 carbon atoms. Suitable alkenylradicals include without limitation ethenyl, propenyl (e.g.,prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), andprop-2-en-2-yl), buten-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl,but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl,hexen-1-yl, 3-hydroxyhexen-1-yl, hepten-1-yl, and octen-1-yl, and thelike. An alkenyl radical may be optionally substituted similar to alkyl.

In aspects of the invention, “substituted alkenyl” includes an alkenylgroup substituted by, for example, one to three substituents, preferablyone to two substituents, such as alkyl, alkoxy, haloalkoxy, alkylalkoxy,haloalkoxyalkyl, alkanoyl, alkanoyloxy, cycloalkyl, cycloalkoxy, acyl,acylamino, acyloxy, amino, alkylamino, alkanoylamino, aminoacyl,aminoacyloxy, cyano, halogen, hydroxyl, carboxyl, carboxylalkyl,carbamyl, keto, thioketo, thiol, alkylthio, sulfonyl, sulfonamido,thioalkoxy, aryl, nitro, and the like.

As used herein, the term “alkynyl” refers to an unsaturated, branched orstraight-chain hydrocarbon radical comprising one or more triple bonds.An alkynyl radical may contain about 1 to 20, 1 to 15, or 2 to 10 carbonatoms, particularly about 3 to 8 carbon atoms and more particularlyabout 3 to 6 carbon atoms. Suitable alkynyl radicals include withoutlimitation ethynyl, such as prop-1-yn-1-yl and prop-2-yn-1-yl, butynylssuch as but-1-yn-1-yl, but-1-yn-3-yl, and but-3-yn-1-yl, pentynyls suchas pentyn-1-yl, pentyn-2-yl, 4-methoxypentyn-2-yl, and3-methylbutyn-1-yl, hexynyls such as hexyn-1-yl, hexyn-2-yl, hexyn-3-yl,and 3,3-dimethylbutyn-1-yl radicals and the like. In aspects of theinvention, alkenyl groups include ethenyl (—CH═CH₂), n-propenyl(—CH₂CH═CH₂), iso-propenyl (—C(CH₃)═CH₂), and the like. An alkynyl maybe optionally substituted similar to alkyl. The term “cycloalkynyl”refers to cyclic alkynyl groups.

In aspects of the invention, “substituted alkynyl” includes an alkynylgroup substituted by, for example, a substituent, such as, alkyl,alkoxy, alkanoyl, alkanoyloxy, cycloalkyl, cycloalkoxy, acyl, acylamino,acyloxy, amino, alkylamino, alkanoylamino, aminoacyl, aminoacyloxy,cyano, halogen, hydroxyl, carboxyl, carboxylalkyl, carbamyl, keto,thioketo, thiol, alkylthio, sulfonyl, sulfonamido, thioalkoxy, aryl,nitro, and the like.

As used herein the term “alkylene” refers to a linear or branchedradical having from about 1 to 10, 1 to 8, 1 to 6, or 2 to 6 carbonatoms and having attachment points for two or more covalent bonds.Examples of such radicals are methylene, ethylene, propylene, butylene,pentylene, hexylene, ethylidene, methylethylene, and isopropylidene.When an alkenylene radical is present as a substituent on anotherradical it is typically considered to be a single substituent ratherthan a radical formed by two substituents.

As used herein the term “alkenylene” refers to a linear or branchedradical having from about 2 to 10, 2 to 8 or 2 to 6 carbon atoms, atleast one double bond, and having attachment points for two or morecovalent bonds. Examples of alkenylene radicals include 1,1-vinylidene(—CH₂═C—), 1,2-vinylidene (—CH═CH—), and 1,4-butadienyl (—CH═CH—CH═CH—).

As used herein the term “halo” refers to a halogen such as fluorine,chlorine, bromine or iodine atoms.

As used herein the term “hydroxyl” or “hydroxy” refers to an —OH group.

As used herein the term “cyano” refers to a carbon radical having threeof four covalent bonds shared by a nitrogen atom, in particular —C—N. Acyano group may be substituted with substituents described herein.

As used herein the term “alkoxy” refers to a linear or branchedoxy-containing radical having an alkyl portion of one to about tencarbon atoms, such as a methoxy radical, which may be substituted. Inaspects of the invention an alkoxy radical may comprise about 1-10, 1-8,1-6 or 1-3 carbon atoms. In embodiments of the invention, an alkoxyradical comprises about 1-6 carbon atoms and includes a C₁-C₆alkyl-O-radical wherein C₁-C₆ alkyl has the meaning set out herein.Examples of alkoxy radicals include without limitation methoxy, ethoxy,propoxy, butoxy, isopropoxy and tert-butoxy alkyls. An “alkoxy” radicalmay optionally be substituted with one or more substitutents disclosedherein including alkyl atoms to provide “alkylalkoxy” radicals; haloatoms, such as fluoro, chloro or bromo, to provide “haloalkoxy” radicals(e.g. fluoromethoxy, chloromethoxy, trifluoromethoxy, difluoromethoxy,trifluoroethoxy, fluoroethoxy, tetrafluoroethoxy, pentafluoroethoxy, andfluoropropox) and “haloalkoxyalkyl” radicals (e.g. fluoromethoxymethyl,chloromethoxyethyl, trifluoromethoxymethyl, difluoromethoxyethyl, andtrifluoroethoxymethyl).

As used herein the term “alkenyloxy” refers to linear or branchedoxy-containing radicals having an alkenyl portion of about 2 to 10carbon atoms, such as an ethenyloxy or propenyloxy radical. Analkenyloxy radical may be a “lower alkenyloxy” radical having about 2 to6 carbon atoms. Examples of alkenyloxy radicals include withoutlimitation ethenyloxy, propenyloxy, butenyloxy, and isopropenyloxyalkyls. An “alkenyloxy” radical may be substituted with one or moresubstitutents disclosed herein including halo atoms, such as fluoro,chloro or bromo, to provide “haloalkenyloxy” radicals (e.g.trifluoroethenyloxy, fluoroethenyloxy, difluoroethenyloxy, andfluoropropenyloxy).

A “carbocylic” includes radicals derived from a saturated orunsaturated, substituted or unsubstituted 5 to 14 member organic nucleuswhose ring forming atoms (other than hydrogen) are solely carbon.Examples of carbocyclic radicals are cycloalkyl, cycloalkenyl, aryl, inparticular phenyl, naphthyl, norbornanyl, bicycloheptadienyl, toluoyl,xylenyl, indenyl, stilbenzyl, terphenylyl, diphenylethylenyl,phenylcyclohexyl, acenapththylenyl, anthracenyl, biphenyl, bibenzylyl,and related bibenzylyl homologs, octahydronaphthyl, tetrahydronaphthyl,octahydroquinolinyl, dimethoxytetrahydronaphthyl and the like.

As used herein, the term “cycloalkyl” refers to radicals having fromabout 3 to 15, 3 to 10, 3 to 8, or 3 to 6 carbon atoms and containingone, two, three, or four rings wherein such rings may be attached in apendant manner or may be fused. In aspects of the invention,“cycloalkyl” refers to an optionally substituted, saturated hydrocarbonring system containing 1 to 2 rings and 3 to 7 carbons per ring whichmay be further fused with an unsaturated C₃-C₇ carbocylic ring. Examplesof cycloalkyl groups include single ring structures such as cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl,cyclononyl, cyclodecyl, cyclododecyl, and the like, or multiple ringstructures such as adamantanyl, and the like. In certain aspects of theinvention the cycloalkyl radicals are “lower cycloalkyl” radicals havingfrom about 3 to 10, 3 to 8, 3 to 6, or 3 to 4 carbon atoms, inparticular cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl andcycloheptyl. The term “cycloalkyl” also embraces radicals wherecycloalkyl radicals are fused with aryl radicals or heterocyclylradicals. A cycloalkyl radical may be optionally substituted with groupsas disclosed herein.

In aspects of the invention, “substituted cycloalkyl” includescycloalkyl groups having from 1 to 5 (in particular 1 to 3) substituentsincluding without limitation alkyl, alkenyl, alkoxy, cycloalkyl,substituted cycloalkyl, acyl, acylamino, acyloxy, amino, aminoacyl,aminoacyloxy, oxyacylamino, cyano, halogen, hydroxyl, carboxyl,carboxylalkyl, keto, thioketo, thiol, thioalkoxy, aryl, aryloxy,heteroaryl, heteroaryloxy, hydroxyamino, alkoxyamino, and nitro.

As used herein in respect to certain aspects of the invention, the term“cycloaliphatic” refers to a cycloalkane possessing less than 8 carbonsor a fused ring system consisting of no more than three fusedcycloaliphatic rings. Examples of such groups include, but are notlimited to, decalin and the like.

As used herein in respect to certain aspects of the invention, the term“substituted cycloaliphatic” refers to a cycloalkane possessing lessthan 8 carbons or a fused ring system consisting of no more than threefused rings, and where at least one of the aliphatic hydrogen atoms hasbeen replaced by a halogen, a nitro, a thio, an amino, a hydroxy, aketone, an aldehyde, an ester, an amide, a lower aliphatic, asubstituted lower aliphatic, or a ring (aryl, substituted aryl,cycloaliphatic, or substituted cycloaliphatic). Examples of such groupsinclude, but are not limited to, 1-chlorodecalyl and the like.

A used herein, the term “cycloalkenyl” refers to radicals comprisingabout 4 to 16, 2 to 15, 2 to 10, 2 to 8, 4 to 10, 3 to 8, 3 to 7, 3 to6, or 4 to 6 carbon atoms, one or more carbon-carbon double bonds, andone, two, three, or four rings wherein such rings may be attached in apendant manner or may be fused. In certain aspects of the invention thecycloalkenyl radicals are “lower cycloalkenyl” radicals having three toseven carbon atoms. Examples of cycloalkenyl radicals include withoutlimitation cyclobutenyl, cyclopentenyl, cyclohexenyl and cycloheptenyl.A cycloalkenyl radical may be optionally substituted with groups asdisclosed herein, in particular 1, 2, or 3 substituents which may be thesame or different.

As used herein the term “cycloalkoxy” refers to cycloalkyl radicals (inparticular, cycloalkyl radicals having 3 to 15, 3 to 8 or 3 to 6 carbonatoms) attached to an oxy radical. Examples of cycloalkoxy radicalsinclude cyclohexoxy and cyclopentoxy. A cycloalkoxy radical may beoptionally substituted with groups as disclosed herein.

As used herein, the term “aryl”, alone or in combination, refers to acarbocyclic aromatic system containing one, two or three rings whereinsuch rings may be attached together in a pendant manner or may be fused.In aspects of the invention an aryl radical comprises 4 to 24 carbonatoms, in particular 4 to 10, 4 to 8, or 4 to 6 carbon atoms.Illustrative “aryl” radicals includes without limitation aromaticradicals such as phenyl, benzyl, naphthyl, indenyl, benzocyclooctenyl,benzocycloheptenyl, pentalenyl, azulenyl, tetrahydronaphthyl, indanyl,biphenyl, acephthylenyl, fluorenyl, phenalenyl, phenanthrenyl, andanthracenyl, preferably phenyl.

An aryl radical may be optionally substituted with groups as disclosedherein, in particular hydroxyl, alkyl, carbonyl, carboxyl, thiol, amino,and/or halo, in particular a substituted aryl includes withoutlimitation arylamine and arylalkylamine.

As used herein in respect to certain aspects of the invention, the term“substituted aryl” includes an aromatic ring, or fused aromatic ringsystem consisting of no more than three fused rings at least one ofwhich is aromatic, and where at least one of the hydrogen atoms on aring carbon has been replaced by a halogen, an amino, a hydroxy, anitro, a thio, an alkyl, a ketone, an aldehyde, an ester, an amide, alower aliphatic, a substituted lower aliphatic, or a ring (aryl,substituted aryl, cycloaliphatic, or substituted cycloaliphatic).Examples of such include, but are not limited to, hydroxyphenyl,chlorophenyl and the like.

In aspects of the invention, an aryl radical may be optionallysubstituted with one to four substituents such as alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, aryl,substituted aryl, aralkyl, halo, trifluoromethoxy, trifluoromethyl,hydroxy, alkoxy, alkanoyl, alkanoyloxy, aryloxy, aralkyloxy, amino,alkylamino, arylamino, aralkylamino, dialkylamino, alkanoylamino, thiol,alkylthio, ureido, nitro, cyano, carboxy, carboxyalkyl, carbamyl,alkoxycarbonyl, alkylthiono, arylthiono, arylsulfonylamine, sulfonicacid, alkysulfonyl, sulfonamido, aryloxy and the like. A substituent maybe further substituted by hydroxy, halo, alkyl, alkoxy, alkenyl,alkynyl, aryl or aralkyl. In aspects of the invention an aryl radical issubstituted with hydroxyl, alkyl, carbonyl, carboxyl, thiol, amino,and/or halo. The term “aralkyl” refers to an aryl or a substituted arylgroup bonded directly through an alkyl group, such as benzyl. Otherparticular examples of substituted aryl radicals include chlorobenyzl,and amino benzyl.

As used herein, the term “aryloxy” refers to aryl radicals, as definedabove, attached to an oxygen atom. Exemplary aryloxy groups includenapthyloxy, quinolyloxy, isoquinolizinyloxy, and the like.

As used herein the term “arylalkoxy,” refers to an aryl group attachedto an alkoxy group. Representative examples of arylalkoxy groupsinclude, but are not limited to, 2-phenylethoxy, 3-naphth-2-ylpropoxy,and 5-phenylpentyloxy.

As used herein, the term “aroyl” refers to aryl radicals, as definedabove, attached to a carbonyl radical as defined herein, includingwithout limitation benzoyl and toluoyl. An aroyl radical may beoptionally substituted with groups as disclosed herein.

As used herein the term “heteroaryl” refers to fully unsaturatedheteroatom-containing ring-shaped aromatic radicals having at least oneheteroatom selected from carbon, nitrogen, sulfur and oxygen. Aheteroaryl radical may contain one, two or three rings and the rings maybe attached in a pendant manner or may be fused. In aspects of theinvention the term refers to fully unsaturated heteroatom-containingring-shaped aromatic radicals having from 3 to 15, 3 to 10, 3 to 8, 5 to15, 5 to 10, or 5 to 8 ring members selected from carbon, nitrogen,sulfur and oxygen, wherein at least one ring atom is a heteroatom.Examples of “heteroaryl” radicals, include without limitation, anunsaturated 5 to 6 membered heteromonocyclyl group containing 1 to 4nitrogen atoms, in particular, pyrrolyl, pyrrolinyl, imidazolyl,pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl,pyrazinyl, pyridazinyl, triazolyl, tetrazolyl and the like; anunsaturated condensed heterocyclic group containing 1 to 5 nitrogenatoms, in particular, indolyl, isoindolyl, indolizinyl, benzimidazolyl,quinolyl, isoquinolyl, indazolyl, quinazolinyl, pteridinyl,quinolizidinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl,phenanthridinyl, acridinyl, phenanthrolinyl, phenazinyl, carbazolyl,purinyl, benzimidazolyl, quinolinyl, isoquinolinyl, benzotriazolyl,tetrazolopyridazinyl and the like; an unsaturated 3 to 6-memberedheteromonocyclic group containing an oxygen atom, in particular,2-furyl, 3-furyl, pyranyl, and the like; an unsaturated 5 to 6-memberedheteromonocyclic group containing a sulfur atom, in particular, thienyl,2-thienyl, 3-thienyl, and the like; unsaturated 5 to 6-memberedheteromonocyclic group containing 1 to 2 oxygen atoms and 1 to 3nitrogen atoms, in particular, furazanyl, benzofurazanyl, oxazolyl,isoxazolyl, and oxadiazolyl; an unsaturated condensed heterocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, in particularbenzoxazolyl, benzoxadiazolyl and the like; an unsaturated 5 to6-membered heteromonocyclic group containing 1 to 2 sulfur atoms and 1to 3 nitrogen atoms, for example, thiazolyl, isothiazolyl, thiadiazolyland the like; an unsaturated condensed heterocyclic group containing 1to 2 sulfur atoms and 1 to 3 nitrogen atoms such as benzothiazolyl,benzothiadiazolyl and the like. The term also includes radicals whereheterocyclic radicals are fused with aryl radicals, in particularbicyclic radicals such as benzofuranyl, benzothiophenyl, phthalazinyl,chromenyl, xanthenyl, and the like. A heteroaryl radical may beoptionally substituted with groups as disclosed herein, for example withan alkyl, amino, halogen, etc., in particular a heteroarylamine.

In aspects of the invention, the term refers to an unsaturated 5 to 6membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, inparticular, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl,3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazolyl, tetrazolyl and the like.

A heteroaryl radical may be optionally substituted with groups disclosedherein, for example with an alkyl, amino, halogen, etc., in particular asubstituted heteroaryl radical is a heteroarylamine.

The term “heterocyclic” refers to saturated and partially saturatedheteroatom-containing ring-shaped radicals having at least oneheteroatom selected from carbon, nitrogen, sulfur and oxygen. Aheterocylic radical may contain one, two or three rings wherein suchrings may be attached in a pendant manner or may be fused. In an aspect,the term refers to a saturated and partially saturatedheteroatom-containing ring-shaped radicals having from about 3 to 15, 3to 10, 5 to 15, 5 to 10, or 3 to 8 ring members selected from carbon,nitrogen, sulfur and oxygen, wherein at least one ring atom is aheteroatom. Examplary saturated heterocyclic radicals include withoutlimitiation a saturated 3 to 6-membered heteromonocylic group containing1 to 4 nitrogen atoms [e.g. pyrrolidinyl, imidazolidinyl, andpiperazinyl]; a saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.morpholinyl; sydnonyl]; and, a saturated 3 to 6-memberedheteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms [e.g., thiazolidinyl] etc. Examples of partiallysaturated heterocyclyl radicals include without limitationdihydrothiophene, dihydropyranyl, dihydrofuranyl and dihydrothiazolyl.Illustrative heterocyclic radicals include without limitationaziridinyl, azetidinyl, 2-pyrrolinyl, 3-pyrrolinyl, pyrrolidinyl,azepinyl, 1,3-dioxolanyl, 2H-pyranyl, 4H-pyranyl, piperidinyl,1,4-dioxanyl, morpholinyl, pyrazolinyl, 1,4-dithianyl, thiomorpholinyl,1,2,3,6-tetrahydropyridinyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydrothiopyranyl,thioxanyl, indolinyl, 2H-pyranyl, 4H-pyranyl, dioxanyl, 1,3-dioxolanyl,pyrazolinyl, dihydropyranyl, dihydrothienyl, dihydrofuranyl,pyrazolidinyl, imidazolinyl, imidazolidinyl, 3H-indolyl, quinuclidinyl,quinolizinyl, and the like. In certain compounds of the formula II, whenR⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ arehydrogen, R¹¹ cannot be piperidinyl.

As used herein in respect to certain aspects of the invention, the term“heterocyclic” refers to a cycloalkane and/or an aryl ring system,possessing less than 8 carbons, or a fused ring system consisting of nomore than three fused rings, where at least one of the ring carbon atomsis replaced by oxygen, nitrogen or sulfur. Examples of such groupsinclude, but are not limited to, morpholino and the like.

As used herein in respect to certain aspects of the invention, the term“substituted heterocyclic” refers to a cycloalkane and/or an aryl ringsystem, possessing less than 8 carbons, or a fused ring systemconsisting of no more than three fused rings, where at least one of thering carbon atoms is replaced by oxygen, nitrogen or sulfur, and whereat least one of the aliphatic hydrogen atoms has been replaced by ahalogen, hydroxy, a thio, nitro, an amino, a ketone, an aldehyde, anester, an amide, a lower aliphatic, a substituted lower aliphatic, or aring (aryl, substituted aryl, cycloaliphatic, or substitutedcycloaliphatic). Examples of such groups include, but are not limited to2-chloropyranyl.

The foregoing heteroaryl and heterocyclic groups may be C-attached orN-attached (where such is possible).

As used herein the term “sulfonyl”, used alone or linked to other termssuch as alkylsulfonyl or arylsulfonyl, refers to the divalent radicals—SO₂—. In aspects of the invention, the sulfonyl group may be attachedto a substituted or unsubstituted hydroxyl, alkyl group, ether group,alkenyl group, alkynyl group, aryl group, cycloalkyl group, cycloalkenylgroup, cycloalkynyl group, heterocyclic group, carbohydrate, peptide, orpeptide derivative.

The term “sulfinyl”, used alone or linked to other terms such asalkylsulfinyl (i.e.—S(O)-alkyl) or arylsulfinyl, refers to the divalentradicals —S(O)—.

The term “sulfonate” is art recognized and includes a group representedby the formula:

wherein R¹⁸ is an electron pair, hydrogen, alkyl, cycloalkyl, aryl,alkenyl, alkynyl, cycloalkenyl, cycloalkynyl, heterocyclic,carbohydrate, peptide, or peptide derivative.

The term “sulfate”, used alone or linked to other terms, is artrecognized and includes a group that can be represented by the formula:

wherein R¹⁹ is an electron pair, hydrogen, alkyl, alkenyl, alkynyl,cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heterocyclic,carbohydrate, peptide or peptide derivative.

The term “sulfoxide” refers to the radical —S═O.

As used herein the term “amino”, alone or in combination, refers to aradical where a nitrogen atom (N) is bonded to three substituents beingany combination of hydrogen, hydroxyl, alkyl, cycloalkyl, alkenyl,alkynyl, aryl, silyl, heterocyclic, or heteroaryl which may or may notbe substituted. Generally an “amino group” has the general chemicalformula —NR²⁰R²¹ where R²⁰ and R²¹ can be any combination of hydrogen,hydroxyl, alkyl, cycloalkyl, alkoxy, alkenyl, alkynyl, aryl, carbonylcarboxyl, amino, silyl, heteroaryl, or heterocyclic which may or may notbe substituted. Optionally one substituent on the nitrogen atom may be ahydroxyl group (—OH) to provide an amine known as a hydroxylamine.Illustrative examples of amino groups are amino (—NH₂), alkylamino,acylamino, cycloamino, acycloalkylamino, arylamino, arylalkylamino, andlower alkylsilylamino, in particular methylamino, ethylamino,dimethylamino, 2-propylamino, butylamino, isobutylamino,cyclopropylamino, benzylamino, allylamino, hydroxylamino,cyclohexylamino, piperidinyl, hydrazinyl, benzylamino,diphenylmethylamino, tritylamino, trimethylsilylamino, anddimethyl-tert.-butylsilylamino, which may or may not be substituted.

As used herein the term “thiol” means —SH. A thiol may be substitutedwith a substituent disclosed herein, in particular alkyl (thioalkyl),aryl (thioaryl), alkoxy (thioalkoxy) or carboxyl.

The term “sulfenyl” used alone or linked to other terms such asalkylsulfenyl, refers to the radical —SR²² wherein R²² is not hydrogen.In aspects of the invention R²² is substituted or unsubstituted alkyl,cycloalkyl, alkenyl, alkynyl, aryl, silyl, silylalkyl, heterocyclic,heteroaryl, carbonyl, carbamoyl, alkoxy, or carboxyl.

As used herein, the term “thioalkyl”, alone or in combination, refers toa chemical functional group where a sulfur atom (S) is bonded to analkyl, which may be substituted. Examples of thioalkyl groups arethiomethyl, thioethyl, and thiopropyl. A thioalkyl may be substitutedwith a substituted or unsubstituted carboxyl, aryl, heterocylic,carbonyl, or heterocyclic.

As used herein the term “thioaryl”, alone or in combination, refers to achemical functional group where a sulfur atom (S) is bonded to an arylgroup with the general chemical formula —SR²³ where R²³ is aryl whichmay be substituted. Illustrative examples of thioaryl groups andsubstituted thioaryl groups are thiophenyl, chlorothiophenyl,para-chlorothiophenyl, thiobenzyl, 4-methoxy-thiophenyl,4-nitro-thiophenyl, and para-nitrothiobenzyl.

As used herein the term “thioalkoxy”, alone or in combination, refers toa chemical functional group where a sulfur atom (S) is bonded to analkoxy group with the general chemical formula —SR²⁴ where R²⁴ is analkoxy group which may be substituted. A “thioalkoxy group” may have 1-6carbon atoms i.e. a —S—(O)—C₁-C₆ alkyl group wherein C₁-C₆ alkyl havethe meaning as defined above. Illustrative examples of a straight orbranched thioalkoxy group or radical having from 1 to 6 carbon atoms,also known as a C₁-C₆ thioalkoxy, include thiomethoxy and thioethoxy.

A thiol may be substituted with a substituted or unsubstitutedheteroaryl or heterocyclic, in particular a substituted or unsubstitutedsaturated 3 to 6-membered heteromonocylic group containing 1 to 4nitrogen atoms [e.g. pyrrolidinyl, imidazolidinyl, piperidinyl, andpiperazinyl] or a saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.morpholinyl; sydnonyl], especially a substituted morpholinyl orpiperidinyl.

As used herein, the term “carbonyl” refers to a carbon radical havingtwo of the four covalent bonds shared with an oxygen atom.

As used herein, the term “carboxyl”, alone or in combination, refers to—C(O)OR²⁵— or —C(═O)OR²⁵ wherein R²⁵ is hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, amino, thiol, aryl, heteroaryl,thioalkyl, thioaryl, thioalkoxy, a heteroaryl, or a heterocyclic, whichmay optionally be substituted. In aspects of the invention, the carboxylgroups are in an esterified form and may contain as an esterifying grouplower alkyl groups. In particular aspects of the invention, —C(O)OR²⁵provides an ester or an amino acid derivative. An esterified form isalso particularly referred to herein as a “carboxylic ester”. In aspectsof the invention a “carboxyl” may be substituted, in particularsubstituted with alkyl which is optionally substituted with one or moreof amino, amine, halo, alkylamino, aryl, carboxyl, or a heterocyclic.Examples of carboxyl groups are methoxycarbonyl, butoxycarbonyl,tert.alkoxycarbonyl such as tert.butoxycarbonyl, arylmethyoxycarbonylhaving one or two aryl radicals including without limitation phenyloptionally substituted by for example lower alkyl, lower alkoxy,hydroxyl, halo, and/or nitro, such as benzyloxycarbonyl,methoxybenzyloxycarbonyl, diphenylmethoxycarbonyl,2-bromoethoxycarbonyl, 2-iodoethoxycarbonyltert.butylcarbonyl,4-nitrobenzyloxycarbonyl, diphenylmethoxy-carbonyl, benzhydroxycarbonyl,di-(4-methoxyphenyl-methoxycarbonyl, 2-bromoethoxycarbonyl,2-iodoethoxycarbonyl, 2-trimethylsilylethoxycarbonyl, or2-triphenylsilylethoxycarbonyl. Additional carboxyl groups in esterifiedform are silyloxycarbonyl groups including organic silyloxycarbonyl. Thesilicon substituent in such compounds may be substituted with loweralkyl (e.g. methyl), alkoxy (e.g. methoxy), and/or halo (e.g. chlorine).Examples of silicon substituents include trimethylsilyl anddimethyltert.butylsilyl. In aspects of the invention, the carboxyl groupmay be an alkoxy carbonyl, in particular methoxy carbonyl, ethoxycarbonyl, isopropoxy carbonyl, t-butoxycarbonyl, t-pentyloxycarbonyl, orheptyloxy carbonyl, especially methoxy carbonyl or ethoxy carbonyl.

As used herein, the term “carbamoyl”, alone or in combination, refers toamino, monoalkylamino, dialkylamino, monocycloalkylamino,alkylcycloalkylamino, and dicycloalkylamino radicals, attached to one oftwo unshared bonds in a carbonyl group.

As used herein, the term “carboxamide” refers to the group —CONH—.

As used herein, the term “nitro” means —NO₂—.

As used herein, the term “acyl”, alone or in combination, means acarbonyl or thiocarbonyl group bonded to a radical selected from, forexample, optionally substituted, hydrido, alkyl (e.g. haloalkyl),alkenyl, alkynyl, alkoxy (“acyloxy” including acetyloxy, butyryloxy,iso-valeryloxy, phenylacetyloxy, benzoyloxy, p-methoxybenzoyloxy, andsubstituted acyloxy such as alkoxyalkyl and haloalkoxy), aryl, halo,heterocyclyl, heteroaryl, sulfinyl (e.g. alkylsulfinylalkyl), sulfonyl(e.g. alkylsulfonylalkyl), cycloalkyl, cycloalkenyl, thioalkyl,thioaryl, amino (e.g alkylamino or dialkylamino), and aralkoxy.Illustrative examples of “acyl” radicals are formyl, acetyl,2-chloroacetyl, 2-bromacetyl, benzoyl, trifluoroacetyl, phthaloyl,malonyl, nicotinyl, and the like.

In aspects of the invention, “acyl” refers to a group —C(O)R²⁶, whereR²⁶ is hydrogen, alkyl, cycloalkyl, cycloheteroalkyl, aryl, arylalkyl,heteroalkyl, heteroaryl, and heteroarylalkyl. Examples include, but arenot limited to formyl, acetyl, cyclohexylcarbonyl,cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl and the like.

As used herein the term “phosphonate” refers to a C—PO(OH)₂ orC—PO(OR²⁷)₂ group wherein R²⁷ is alkyl or aryl which may be substituted.

As used herein, “ureido” refers to the group “—NHCONH—”. A ureidoradical includes an alkylureido comprising a ureido substituted with analkyl, in particular a lower alkyl attached to the terminal nitrogen ofthe ureido group. Examples of an alkylureido include without limitationN′-methylureido, N′-ethylureido, N′-n-propylureido, N′-i-propylureidoand the like. A ureido radical also includes a N′,N′-dialkylureido groupcontaining a radical —NHCON where the terminal nitrogen is attached totwo optionally substituted radicals including alkyl, aryl, heterocylic,and heteroaryl.

The terms used herein for radicals including “alkyl”, “alkoxy”,“alkenyl”, “alkynyl”, “hydroxyl” etc. refer to both unsubstituted andsubstituted radicals. The term “substituted,” as used herein, means thatany one or more moiety on a designated atom (e.g., hydrogen) is replacedwith a selection from a group disclosed herein, provided that thedesignated atom's normal valency is not exceeded, and that thesubstitution results in a stable compound. Combinations of substituentsand/or radicals are permissible only if such combinations result instable compounds. “Stable compound” refers to a compound that issufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

A functional group or ring of a compound of the formula I, II or III maybe modified with, or a radical in a compound of the formula I, II or IIImay be substituted with one or more groups or substituents apparent to aperson skilled in the art including without limitation alkyl, alkoxy,alkenyl, alkynyl, alkanoyl, alkylene, alkenylene, hydroxyalkyl,haloalkyl, haloalkylene, haloalkenyl, alkoxy, alkenyloxy,alkenyloxyalkyl, alkoxyalkyl, aryl, alkylaryl, haloalkoxy,haloalkenyloxy, heterocyclic, heteroaryl, alkylsulfonyl, sulfinyl,sulfonyl, sulfenyl, alkylsulfinyl, aralkyl, heteroaralkyl, cycloalkyl,cycloalkenyl, cycloalkoxy, cycloalkenyloxy, amino, oxy, halo, azido,thio, ═O, ═S, cyano, hydroxyl, phosphonato, phosphinato, thioalkyl,alkylamino, arylamino, arylsulfonyl, alkylcarbonyl, arylcarbonyl,heteroarylcarbonyl, heteroarylsulfinyl, heteroarylsulfonyl,heteroarylamino, heteroaryloxy, heteroaryloxylalkyl, arylacetamidoyl,aryloxy, aroyl, aralkanoyl, aralkoxy, aryloxyalkyl, haloaryloxyalkyl,heteroaroyl, heteroaralkanoyl, heteroaralkoxy, heteroaralkoxyalkyl,thioaryl, arylthioalkyl, alkoxyalkyl, and acyl groups. These groups orsubstitutents may themselves be substituted. Derivative groups that maybe used to modify compounds of the Formula I can also be found in U.S.Patent Application No. 20030176437.

A chemical substituent is “pendant” from a radical if it is bound to anatom of the radical. In this context, the substituent can be pendingfrom a carbon atom of a radical, a carbon atom connected to a carbonatom of the radical by a chain extender, or a heteroatom of the radical.The term “fused” means that a second ring is present (i.e, attached orformed) by having two adjacent atoms in common or shared with the firstring.

A “dosage form” refers to a composition or device comprising a compoundof the formula I, II or III and optionally pharmaceutically acceptablecarrier(s), excipient(s), or vehicles. A dosage form may be an immediaterelease dosage form or a sustained release dosage form.

An “immediate release dosage form” refers to a dosage form which doesnot include a component for sustained release i.e., a component forslowing disintegration or dissolution of an active compound. Thesedosage forms generally rely on the composition of the drug matrix toeffect the rapid release of the active ingredient agent.

By “sustained release dosage form” is meant a dosage form that releasesactive compound for many hours. In an aspect, a sustained dosage formincludes a component for slowing disintegration or dissolution of theactive compound. A dosage form may be a sustained release formulation,engineered with or without an initial delay period. Sustained releasedosage forms may continuously release drug for sustained periods of atleast about 4 hours or more, about 6 hours or more, about 8 hours ormore, about 12 hours or more, about 15 hours or more, or about 20 hoursto 24 hours. A sustained release dosage form can be formulated into avariety of forms, including tablets, lozenges, gelcaps, buccal patches,suspensions, solutions, gels, etc. In aspects of the invention thesustained release form results in administration of a minimum number ofdaily doses.

A “disease” that can be treated and/or prevented using a compound,composition, or method of the invention includes a condition associatedwith or requiring modulation of one or more of inflammation (e.g.neuroinflammation); signaling pathways involved in inflammation (e.g.,neuroinflammation); cell signaling molecule production; activation ofglia or glial activation pathways and responses; proinflammatorycytokines or chemokines (e.g., interleukin (IL), in particular IL-1β) ortumor necrosis factor (TNF, in particular TNFα); activation ofastrocytes or astrocyte activation pathways and responses; activation ofmicroglia or microglial activation pathways and responses; oxidativestress-related responses such as nitric oxide synthase production andnitric oxide accumulation; acute phase proteins; loss of synaptophysinand/or 95; components of the complement cascade; loss or reduction ofsynaptic function; protein kinase activity (e.g., death associatedprotein kinase activity); cell damage (e.g., neuronal cell damage); celldeath (e.g., neuronal cell death); amyloid β deposition of amyloidplaques; and behavioral deficits.

In particular a disease is a neurological disease or condition includingwithout limitation, dementing disorder, a neurodegenerative disorder, aCNS demyelinating disorder, a pain disorder, an autoimmune disorder, ora peripheral inflammatory disease.

A disease may be characterized by an inflammatory process due to thepresence of macrophages activated by an amyloidogenic protein orpeptide. Thus, a method of the invention may involve inhibitingmacrophage activation and/or inhibiting an inflammatory process. Amethod may comprise decreasing, slowing, ameliorating, or reversing thecourse or degree of macrophage invasion or inflammation in a patient.

Examples of diseases that can be treated and/or prevented using thecompounds, compositions and methods of the invention include Alzheimer'sdisease and related disorders, presenile and senile forms; amyloidangiopathy; mild cognitive impairment; Alzheimer's disease-relateddementia (e.g., vascular dementia or Alzheimer dementia); AIDS relateddementia, tauopathies (e.g., argyrophilic grain dementia, corticobasaldegeneration, dementia pugilistica, diffuse neurofibrillary tangles withcalcification, frontotemporal dementia with parkinsonism, Prion-relateddisease, Hallervorden-Spatz disease, myotonic dystrophy, Niemann-Pickdisease type C, non-Guamanian Motor Neuron disease with neurofibrillarytangles, Pick's disease, postencephalitic parkinsonism, cerebral amyloidangiopathy, progressive subcortical gliosis, progressive supranuclearpalsy, subacute sclerosing panencephalitis, and tangle only dementia),alpha-synucleinopathy (e.g., dementia with Lewy bodies, multiple systematrophy with glial cytoplasmic inclusions), multiple system atrophies,Shy-Drager syndrome, spinocerebellar ataxia (e.g., DRPLA orMachado-Joseph Disease); striatonigral degeneration,olivopontocerebellar atrophy, neurodegeneration with brain ironaccumulation type I, olfactory dysfunction, and amyotrophic lateralsclerosis); Parkinson's disease (e.g., familial or non-familial);Amyotrophic Lateral Sclerosis; Spastic paraplegia (e.g., associated withdefective function of chaperones and/or triple A proteins); Huntington'sDisease, spinocerebellar ataxia, Freidrich's Ataxia; cerebrovasculardiseases including stroke, hypoxia, ischemia, infarction, intracerebralhemorrhage; traumatic brain injury; Down's syndrome; head trauma withpost-traumatic accumulation of amyloid beta peptide; Familial BritishDementia; Familial Danish Dementia; Presenile Dementia with SpasticAtaxia; Cerebral Amyloid Angiopathy, British Type; Presenile DementiaWith Spastic Ataxia Cerebral Amyloid Angiopathy, Danish Type; Familialencephalopathy with neuroserpin inclusion bodies (FENIB); AmyloidPolyneuropathy (e.g., senile amyloid polyneuropathy or systemicAmyloidosis); Inclusion Body myositis due to amyloid beta peptide;Familial and Finnish Type Amyloidosis; Systemic amyloidosis associatedwith multiple myeloma; Familial Mediterranean Fever; multiple sclerosis,optic neuritis; Guillain-Barre Syndrome; chronic inflammatorydemyelinating polyneuropathy; chronic infections and inflammations;acute disseminated encephalomyelitis (ADEM); autoimmune inner eardisease (AIED); diabetes; myocardial ischemia and other cardiovasculardisorders; pancreatitis; gout; inflammatory bowel disease; ulcerativecolitis, Crohn's disease, rheumatoid arthritis, osteoarthritis;artheriosclerosis, inflammatory aortic aneurysm; asthma; adultrespiratory distress syndrome; restenosis; ischemia/reperfusion injury;glomerulonephritis; sacoidosis cancer; restenosis; rheumatic fever;systemic lupus erythematosus; Reiter's syndrome; psoriatic arthritis;ankylosing spondylitis; coxarthritis; pelvic inflammatory disease;osteomyelitis; adhesive capsulitis; oligoarthritis; periarthritis;polyarthritis; psoriasis; Still's disease; synovitis; inflammatorydermatosis; and, wound healing.

In aspects of the invention, the disease is Alzheimer's disease,vascular dementia, dementia associated with Parkinson's disease,visuospatial deficits, Williams syndrome, encephalitis, meningitis,fetal alcohol syndrome, Korsakoffs syndrome, anoxic brain injury,cardiopulmonary resuscitation injuries, diabetes, Sjogren's syndrome,strokes, ocular diseases such as cataracts and macular degeneration,sleep disorders, and cognitive impairments caused by high cholesterollevels.

In aspects of the invention, a compound, composition, or methoddisclosed herein may be utilized to prevent and/or treat a diseaseinvolving neuroinflammation (i.e., neuroinflammatory disease).Neuroinflammation is a characteristic feature of disease pathology andprogression in a diverse array of neurodegenerative disorders that areincreasing in their societal impact (for a recent review, see, e.g.,Prusiner, S. B. (2001) New Engl. J. Med. 344, 1516-1526). Theseneuroinflammation-related disorders include Alzheimer's disease (AD),amyotrophic lateral sclerosis, autoimmune disorders, priori diseases,stroke and traumatic brain injury. Neuroinflammation is brought about byglial cell (e.g., astrocytes and microglia) activation, which normallyserves a beneficial role as part of an organism's homeostatic responseto injury or developmental change. However, disregulation of thisprocess through chronic or excessive activation of glia contributes tothe disease process through the increased production of proinflammatorycytokines and chemokines, oxidative stress-related enzymes, acute phaseproteins, and various components of the complement cascades. (See, e.g.,Akiyama et al., (2000) Neurobiol. Aging 21, 383-421). The direct linkageof glial activation to pathology that is a hallmark of diseaseunderscores the importance of understanding the signal transductionpathways that mediate these critical glial cellular responses and thediscovery of cell permeable ligands that can modulate these diseaserelevant pathways.

In certain selected aspects of the invention, the disease is aneurodegenerative disease or neurodegenerative disorder including suchdiseases and impairments as Alzheimer's disease, dementia, MCI,Huntington's disease, Parkinson's disease, amyotrophic lateralsclerosis, and other similar diseases and disorders disclosed herein.

For Alzheimer's disease (AD) in particular, the deposition of β-amyloid(Aβ) and neurofibrillary tangles are associated with glial activation,neuronal loss and cognitive decline. On a molecular level, Alzheimer'sdisease is characterized by; increased expression of nitric oxidesynthase (NOS) in glial cells surrounding amyloid plaques;neuropathological evidence of peroxynitrite-mediated neuronal damage;and nitric oxide (NO) overproduction involved in Aβ-induced braindysfunction. NOSH (iNOS) is induced as part of the glial activationresponse and is an oxidative stress-related enzyme that generates NO.When NO is present in high levels along with superoxide, the highlyreactive NO-derived molecule peroxynitrite is generated, leading toneuronal cell death. The pro-inflammatory cytokine IL-1β is alsooverexpressed in activated glia in AD brain and polymorphisms in IL-1βgenes are associated with an increased risk of early onset sporadic AD(See, e.g., Du et al., (2000) Neurology 55, 480-483). IL-1β can alsoinfluence amyloid plaque development and is involved in additional glialinflammatory and neuronal dysfunction responses (See, e.g., Griffin, etal., (1998) Brain Pathol. 8, 65-72; and Sheng, et al., (1996) Neurobiol.Aging 17, 761-766). Therefore, because glial activation and specificglial products are associated with neurodegenerative disorders (e.g.,Alzheimer's disease), the compounds and compositions disclosed hereinthat are capable of modulating cell signaling pathways (e.g., glialactivation pathways) will have particular application in the treatmentand prevention of inflammatory disease.

In aspects of the invention, a compound, composition, or methoddisclosed herein may be utilized to prevent and/or treat a diseaseinvolving disregulation of protein kinase signaling. Disregulation ofprotein kinase signaling often accompanies disregulation of cellsignaling pathways (e.g., glial cell activation pathways). Proteinkinases are a large family of proteins that play a central role inregulating a number of cellular functions including cell growth,differentiation and death. There are thought to be more than 500 proteinkinases and 130 protein phosphatases exerting tight control on proteinphosphorylation. Each protein kinase transfers the γ-phosphate of ATP toa specific residue(s) of a protein substrate. Protein kinases can befurther categorized as tyrosine, serine/threonine or dual specific basedon acceptor residue. Examples of serine/threonine kinases include MAPkinase, MAPK kinase (MEK), Akt/PKB, Jun kinase (INK), CDKs, proteinkinase A (PRA), protein kinase C(PKC), and calmodulin (CaM)-dependentkinases (CaMKs). Disregulated protein kinase activity (e.g., hyper- orhypo-active) leads to abnormal protein phosphorylation, underlying agreat number of diseases including diabetes, rheumatoid arthritis,inflammation, hypertension, and proliferative diseases such as cancer.Therefore, because aberrant kinase activity is associated withinflammatory disease (e.g., neurodegenerative disorders like Alzheimer'sdisease), the compounds and compositions that are disclosed herein thatare capable of modulating kinases involved in cell signaling pathwayswill have particular application for treatment and prevention ofinflammatory disease.

Diseases that may also be treated and/or prevented according to theinvention include Demyelinating Diseases. “Demyelinating Diseases”refers to diseases in which myelin is the primary target. These diseasescan be divided into two groups: Acquired Diseases and HereditaryMetabolic Disorders. Acquired Demyelinating Diseases include Multiplesclerosis (MS) including its alternating relapsing/remitting phases.Hereditary Metabolic Disorders includes the leukodystrophies such asmetachromatic leukodystrophy, Refsum's disease, adrenoleukodystrophy,Krabbe's disease, phenylketonuria, Canavan disease, Pelizaeus-Merzbacherdisease and Alexander's disease.

Diseases that may also be treated and/or prevented according to theinvention include “Demyelinating Conditions”. The term refers toconditions that result in deficient myelination. Such conditionsinclude, but are not limited to, Spinal Cord Injury, Traumatic BrainInjury and Stroke.

The term “Spinal Cord Injury (SCI)” refers to an injury to the spinalcord which results in loss of function such as mobility or feeling.

The term “Traumatic Brain Injury (TBI)” refers to an injury whichresults in damage to the brain. A head injury may be a closed headinjury or penetrating head injury. A closed head injury may occur whenthe head is hit by a blunt object causing the brain to interact with thehard bony surface inside the skull. A closed head injury may also occurwithout direct external trauma to the head if the brain undergoes arapid forward or backward movement, (e.g. whiplash). A penetrating headinjury may occur when a fast moving object such as a bullet pierces theskull. A closed or penetrating head injury may result in localized andwidespread, or diffuse, damage to the brain which may manifest as memoryloss, emotional disturbances, motor difficulties, including paralysis,damage to the senses, and death. The term also includes secondary damagethat follows an injury including swelling and fluid buildup and theaccumulation of substances toxic to surrounding neurons such as theneurotransmitter glutamate.

The term “Stroke” refers to a sudden loss of brain function caused bythe interruption of the flow of blood to the brain (an ischemic stroke)or the rupture of blood vessels in the brain (a hemorrhagic stroke). Theinterruption of the blood flow or the rupture of blood vessels causesneurons in the affected area to die. The term also includes strokerehabilitation which refers to the intervention resulting in the full orpartial recovery of functions that have been lost due to stroke.

A pain disorder may also be treated and/or prevented according to theinvention. A “pain disorder” refers to a disorder or condition involvingpain and includes without limitation acute pain, persistent pain,chronic pain, inflammatory pain, neuropathic pain, neurogenic pain, andchemokine-induced pain. In aspects of the invention, a pain disorderincludes without limitation pain resulting from soft tissue andperipheral damage such as acute trauma; complex regional pain syndromealso referred to as reflex sympathetic dystrophy; postherpeticneuralgia, occipital neuralgia, trigeminal neuralgia, segmental orintercostal neuralgia and other neuralgias; pain associated withosteoarthritis and rheumatoid arthritis; musculo-skeletal pain such aspain associated with strains, sprains and trauma such as broken bones;spinal pain, central nervous system pain such as pain due to spinal cordor brain stem damage; lower back pain, sciatica, dental pain, myofascialpain syndromes, episiotomy pain, gout pain, and pain resulting fromburns; deep and visceral pain, such as heart pain; muscle pain, eyepain, inflammatory pain, orofacial pain, for example, odontalgia;abdominal pain, and gynecological pain, for example, dysmenorrhoea,labour pain and pain associated with endometriosis; somatogenic pain;pain associated with nerve and root damage, such as pain associated withperipheral nerve disorders, for example, nerve entrapment, brachialplexus avulsions, and peripheral neuropathies; pain associated with limbamputation, tic douloureux, neuroma, or vasculitis; diabetic neuropathy,chemotherapy-induced-neuropathy, acute herpetic and postherpeticneuralgia; atypical facial pain, nerve root damage, neuropathic lowerback pain, HIV related neuropathic pain, cancer related neuropathicpain, diabetes related neuropathic pain and arachnoiditis, trigeminalneuralgia, occipital neuralgia, segmental or intercostal neuralgia, HIVrelated neuralgias and AIDS related neuralgias and other neuralgias;allodynia, hyperalgesia, idiopathic pain, pain caused by chemotherapy;occipital neuralgia, psychogenic pain, brachial plexus avulsion, painassociated with restless legs syndrome; pain associated with gallstones;pain caused by chronic alcoholism or hypothyroidism or uremia or vitamindeficiencies; neuropathic and non-neuropathic pain associated withcarcinoma, often referred to as cancer pain, phantom limb pain,functional abdominal pain, headache, including migraine with aura,migraine without aura and other vascular headaches, acute or chronictension headache, sinus headache and cluster headache; temperomandibularpain and maxillary sinus pain; pain resulting from ankylosingspondylitis and gout; pain caused by increased bladder contractions;pain associated with gastrointestinal (GI) disorders, disorders causedby helicobacter pylori and diseases of the GI tract such as gastritis,proctitis, gastroduodenal ulcers, peptic ulcers, dyspepsia, disordersassociated with the neuronal control of viscera, ulcerative colitis,chronic pancreatitis, Crohn's disease and emesis; post operative pain,scar pain, and chronic non-neuropathic pain such as pain associated withHIV, anthralgia and myalgia, vasculitis and fibromyalgia.

The term “Neuropathic pain” refers to pain initiated or caused by aprimary lesion or dysfunction in the nervous system. For the purpose ofthis invention included under this heading or to be treated assynonymous is “Neurogenic Pain” which is defined as pain initiated orcaused by a primary lesion, dysfunction or transitory perturbation inthe peripheral or central nervous system. In aspects, the uses of thepresent invention include central or peripheral neuropathic pain orneurogenic pain. In other aspects, neuropathic pain includes the paincaused by either mononeuropathy or polyneuropathy. Neuropathic pain alsoincludes Chemokine-Induced Pain.

“Peripheral neuropathic pain” refers to a pain initiated or caused by aprimary lesion or dysfunction in the peripheral nervous system and“peripheral neurogenic pain” refers to a pain initiated or caused by aprimary lesion, dysfunction or transitory perturbation in the peripheralnervous system. A peripheral neuropathic pain can be allodynia (i.e., apain due to a stimulus which does not normally provoke pain); causalgia(i.e., a syndrome of sustained burning pain, allodynia and hyperpathiaafter a traumatic nerve lesion, often combined with vasomotor andsudomotor dysfunction and later trophic changes); hyperalgesia (i.e., anincreased response to a stimulus which is normally painful);hyperesthesia (i.e., increased sensitivity to stimulation, excluding thesenses); hyperpathia. (i.e., a painful syndrome characterized by anabnormally painful reaction to a stimulus, especially a repetitivestimulus, as well as an increased threshold); neuritis (i.e.,inflammation of a nerve or nerves); or neuropathy (i.e., a disturbanceof function or pathological change in a nerve). [See IASP,Classification of chronic pain, 2nd Edition, IASP Press (2002), fordetailed definitions of these categories of neuropathic pain andneurogenic pain).

Exemplary types of neuropathic pain include infective (e.g., postherpetic neuralgia and HIV neuropathy), metabolic (e.g., diabeticneuropathy and Fabry's disease), toxic (e.g., from lead orchemotherapy), traumatic/stretch injury (e.g., post incisional, trauma,phantom limb pain, and reflex sympathetic dystrophy/complex regionalpain syndrome/causalgia), and idiopathic (e.g., trigeminal neuralgia/ticdouloureux).

Particular examples of Neuropathic Pain include post-herpetic neuralgia,painful diabetic neuropathy, phantom limb pain, central post-strokepain, HIV neuropathy, Fabry's disease, peripheral neuropathy, trigeminalneuralgia, post incisional neuropathic pain, phantom limb pain, reflexsympathetic dystrophy, causalgia, anesthesia dolorosa, intercoastalneuralgia, post-traumatic localized pain, atypical facial neuralgia painafter tooth extraction and the like, complex regional pain syndrome,neuropathic pain caused by trauma, lead, or chemotherapy, cancer painresistant to narcotic analgesics such as morphine.

Treatment of neuropathic pain may be defined as administration of atherapeutic dose of a compound of the formula I, II or III to reduce andpreferably eliminate pain that results from nerve injury. Treatment ofnerve injury may be defined as administration of a therapeutic dose of acompound of the formula I, II or III to ameliorate injury and toincrease the rate of recovery. An increased rate of recovery is definedas a reduction of indications of pain from peripheral nerve injury, suchas thermal hyperalgesia and mechanical allodynia, more quickly thanwould be accomplished without pharmacological or other medicalintervention.

“Chemokine-Induced Pain” refers to pain that occurs in response, inwhole or in part, to chemokines, in particular pro-inflammatorycytokines (e.g. fractalkine, CCL2, and CCL5). An example ofchemokine-induced pain is arthritic pain.

Compounds and Processes

The invention contemplates the use of isolated and pure, in particular,substantially pure, compounds of the formula I wherein R¹, R⁴, R⁵, R⁶,R⁷, R⁸, R⁹, R¹², R¹³, and R¹⁴ are independently hydrogen, hydroxyl,alkyl, alkenyl, alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy,cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy, aryl, aryloxy,arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino,imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano,halo, sulfate, sulfenyl, sulfinyl, sulfonyl, sulfonate, sulfoxide,silyl, silyloxy, silylalkyl, silylthio, ═O, ═S, phosphonate, ureido,carboxyl, carbonyl, carbamoyl, or carboxamide; and X is optionallysubstituted pyrimidinyl or pyridazinyl, an isomer, a pharmaceuticallyacceptable salt, or derivative thereof.

The invention also contemplates the use of isolated and pure, inparticular, substantially pure, compounds of the formula I wherein R¹,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹², R¹³, and R¹⁴ are independently hydrogen,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxy, C₂-C₆alkenyloxy, C₃-C₁₀ cycloalkyl, C₄-C₁₀cycloalkenyl, C₃-C₁₀cycloalkoxy,C₆-C₁₀aryl, C₆-C₁₀aryloxy, C₆-C₁₀aryl-C₁-C₃alkoxy, C₆-C₁₀aroyl,C₆-C₁₀heteroaryl, C₃-C₁₀heterocyclic, C₁-C₆acyl, C₁-C₆acyloxy, —NH₂,—NHR²⁸, —NR²⁸R²⁸, ═NR²⁸, —S(O)₂R²⁸, —SH, —SO₃H, nitro, cyano, halo,haloalkyl, haloalkoxy, hydroxyalkyl, —CO₂H, —CO₂R²⁸, —NHC(O)R²⁸,—C(O)NR²⁸, —C(O)NHR²⁸, —C(O)NR²⁸R²⁹, —NHS(O)₂R^(X), wherein R²⁸ and R²⁹are independently selected from C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₁₀cycloalkyl, C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀ arylC₁-C₃alkyl, C₆-C₁₀ heteroaryl and C₃-C₁₀heterocyclic, and X ispyrimidinyl or pyridazinyl, an isomer, a pharmaceutically acceptablesalt, or derivative thereof.

The invention further contemplates the use of isolated and pure, inparticular, substantially pure, compounds of the formula II wherein R¹,R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ are independentlyhydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy,aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl,acyloxy, sulfoxide, sulfate, sulfonyl, sulfenyl, sulfinyl, sulfonate,amino, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro,cyano, halo, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S,phosphonate, ureido, carboxyl, carbonyl, carbamoyl, or carboxamide; oran isomer, a pharmaceutically acceptable salt, or derivative thereof.

The invention still further contemplates the use of isolated and pure,in particular, substantially pure, compounds of the formula II whereinR¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, and R¹⁴ areindependently selected from hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy, C₃-C₁₀ cycloalkyl,C₄-C₁₀cycloalkenyl, C₃-C₁₀cycloalkoxy, C₆-C₁₀aryl, C₆-C₁₀aryloxy,C₆-C₁₀aryl-C₁-C₃alkoxy, C₆-C₁₀aroyl, C₆-C₁₀heteroaryl,C₃-C₁₀heterocyclic, C₁-C₆acyl, C₁-C₆acyloxy, —NH₂, —NHR²⁸, —NR²⁸R²⁹,═NR²⁸, —S(O)₂R⁹, —SH, —SO₃H, nitro, cyano, halo, haloalkyl, haloalkoxy,hydroxyalkyl, —CO₂H, —CO₂R²⁸, —NHC(O)R²⁸, —C(O)NH₂, —C(O)NHR^(X),—C(O)NR^(X)R^(X), —NHS(O)₂R^(X), wherein R²⁸ and R²⁹ are independentlyselected from C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₁₀cycloalkyl,C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀ aryl C₁-C₃alkyl, C₆-C₁₀heteroaryl and C₃-C₁₀heterocyclic, or an isomer, a pharmaceuticallyacceptable salt, or derivative thereof.

In aspects of the invention, R¹ in a compound of the formula I or II isalkyl, alkenyl, alkynyl, alkoxy, or cycloalkyl. In certain aspects ofthe invention, R¹ in a compound of the formula I or II is C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxy, or C₃-C₁₀ cycloalkyl. Inembodiments, R¹ is lower alkyl. In another embodiment, R¹ is cyclohexyl.

In aspects of the invention, R¹ in a compound of the formula I or II isaryl, in particular phenyl, benzyl, naphthyl, indenyl,benzocyclooctenyl, benzocycloheptenyl, pentalenyl, azulenyl,tetrahydronaphthyl, indanyl, biphenyl, acephthylenyl, fluorenyl,phenalenyl, phenanthrenyl, and anthracenyl. In aspects of the inventionR¹ is aryl substituted with one or more of hydroxyl, alkyl, carbonyl,carboxyl, thiol, amino, nitro, ketone, aldehyde, ester, amide, loweraliphatic, aryl, cycloalkyl, and halo. In aspects of the invention R¹ ina compound of the formula I or II comprises two fused aromatic rings.

In aspects of the invention, R¹ in a compound of the formula I or II isaryloxy, in particular C₆-C₁₀aryloxy. In embodiments of the invention,R¹ in a compound of the formula I or II is napthyloxy, quinolyloxy,isoquinolizinyloxy, and the like.

In aspects of the invention, R¹ in a compound of the formula I or II isarylalkoxy, in particular C₆-C₁₀aryloxy or C₆-C₁₀aryl-C₁-C₃alkoxy. Inembodiments, R¹ in a compound of the formula I or II is 2-phenylethoxy,3-naphth-2-ylpropoxy, and 5-phenylpentyloxy.

In aspects of the invention, R¹ in a compound of the formula I or II isaroyl, in particular C₆-C₁₀aroyl. In embodiments of the invention R¹ ina compound of the formula I or II is benzoyl or toluoyl.

In aspects of the invention, R¹ in a compound of the formula I or II isa heteroaryl, in particular C₆-C₁₀heteroaryl. In aspects, R¹ in acompound of the formula I or II comprises one or two rings attached in apendant manner or fused. In certain aspects of the invention, R¹ in acompound of the formula I or II is: (a) an unsaturated 5 to 6 memberedheteromonocyclyl group containing 1 to 4 nitrogen atoms, mostparticularly, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl,3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazolyl, tetrazolyl and the like; (b) an unsaturated condensedheterocyclic group containing 1 to 5 nitrogen atoms, in particular,indolyl, isoindolyl, indolizinyl, indazolyl, quinazolinyl, pteridinyl,quinolizidinyl, phthalazinyl, naphthyridinyl, quinoxalinyl, cinnolinyl,phenanthridinyl, acridinyl, phenanthrolinyl, phenazinyl, carbazolyl,purinyl, benzimidazolyl, quinolyl, isoquinolyl, quinolinyl,isoquinolinyl, indazolyl, benzotriazolyl, tetrazolopyridazinyl and thelike; (c) an unsaturated 3 to 6-membered heteromonocyclic groupcontaining an oxygen atom, in particular, 2-furyl, 3-furyl, pyranyl, andthe like; (d) an unsaturated 5 to 6-membered heteromonocyclic groupcontaining a sulfur atom, in particular, thienyl, 2-thienyl, 3-thienyl,and the like; (e) an unsaturated 5 to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, in particular,furazanyl, benzofurazanyl, oxazolyl, isoxazolyl, and oxadiazolyl; (f) anunsaturated condensed heterocyclic group containing 1 to 2 oxygen atomsand 1 to 3 nitrogen atoms, in particular benzoxazolyl, benzoxadiazolyland the like; (g) an unsaturated 5 to 6-membered heteromonocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms, for example,thiazolyl, isothiazolyl, thiadiazolyl and the like; or (h) anunsaturated condensed heterocyclic group containing 1 to 2 sulfur atomsand 1 to 3 nitrogen atoms such as benzothiazolyl, benzothiadiazolyl andthe like.

In certain aspects of the invention, R¹ in a compound of the formula Ior II is a heterocyclic fused with an aryl, in particular benzofuranyl,benzothiophenyl, phthalazinyl, chromenyl, xanthenyl, and the like.

In particular aspects of the invention R¹ in a compound of the formula Ior II is:

wherein R¹⁵, R¹⁶ and R¹⁷ are independently hydrogen, hydroxyl, alkyl,alkenyl, alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl,cycloalkenyl, cycloalkynyl, cycloalkoxy, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino, imino, azido,thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo, sulfoxide,sulfate, sulfonyl, sulfenyl, sulfinyl, sulfonate, silyl, silyloxy,silylalkyl, silylthio, ═O, ═S, phosphonate, ureido, carboxyl, carbonyl,carbamoyl, or carboxamide.

In embodiments of the invention, R¹⁵, R¹⁶ and R¹⁷ are independentlyhydrogen, hydroxyl, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl,C₁-C₆alkoxy, C₂-C₆ alkenyloxy, C₃-C₁₀ cycloalkyl, C₄-C₁₀cycloalkenyl,C₃-C₁₀cycloalkoxy, C₆-C₁₀aryl, C₆-C₁₀aryloxy, C₆-C₁₀aryl-C₁-C₃alkoxy,C₆-C₁₀aroyl, C₆-C₁₀heteroaryl, C₃-C₁₀heterocyclic, C₁-C₆acyl,C₁-C₆acyloxy, —NH₂, —NHR²⁸, —NR²⁸R²⁹, ═NR²⁸, ═O, ═S, nitro, cyano, halo,haloalkyl, haloalkoxy, hydroxyalkyl, —CO₂H, —CO₂R²⁸, —NHC(O)R²⁸,—C(O)NH₂, —C(O)NHR²⁸, —C(O)NR²⁸R²⁹, —NHS(O)₂R²⁸, wherein R²⁸ and R²⁹ areindependently selected from C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₁₀cycloalkyl, C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀ arylC₁-C₃alkyl, C₆-C₁₀ heteroaryl and C₃-C₁₀heterocyclic.

In other particular aspects of the invention a compound of the formulaIII is employed.

wherein R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶ andR¹⁷ are independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl,alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl,cycloalkynyl, cycloalkoxy, aryl, aryloxy, arylalkoxy, aroyl, heteroaryl,heterocyclic, acyl, acyloxy, amino, imino, azido, thiol, thioalkyl,thioalkoxy, thioaryl, nitro, cyano, halo, sulfoxide, sulfate, sulfonyl,sulfenyl, sulfinyl, sulfonate, silyl, silyloxy, silylalkyl, silylthio,═O, ═S, phosphonate, ureido, carboxyl, carbonyl, carbamoyl, orcarboxamide.

In aspects of the invention R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³,R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are independently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, Cl C₆alkoxy, C₂-C₆ alkenyloxy,C₃-C₁₀ cycloalkyl, C₄-C₁₀cycloalkenyl, C₃-C₁₀cycloalkoxy, C₆-C₁₀aryl,C₆-C₁₀aryloxy, C₆-C₁₀aryl-C₁-C₃alkoxy, C₆-C₁₀aroyl, C₆-C₁₀heteroaryl,C₃C₁₀heterocyclic, C₁-C₆acyl, C₁-C₆acyloxy, —NH₂, —NHR²⁸, —NR²⁸R²⁹,═NR²⁸, —S(O)₂R²⁸, —SH, —SO₃H, nitro, cyano, halo, haloalkyl, haloalkoxy,hydroxyalkyl, —CO₂H, —CO₂R²⁸, —NHC(O)R²⁸, —C(O)NH₂, —C(O)NHR²⁸,—C(O)NR²⁸R²⁹, —NHS(O)₂R²⁸, wherein R²⁸ and R²⁹ are independentlyselected from C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₁₀cycloalkyl,C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀ aryl C₁-C₃alkyl, C₆-C₁₀heteroaryl and C₃-C₁₀heterocyclic.

In general, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶,and R¹⁷ in a compound of the formula III cannot all be hydrogen. Inaspects of the invention a compound of the formula III is providedwherein both of R¹⁰ and R¹¹ are not hydrogen. In other aspects of theinvention a compound of the formula II is provided wherein R¹¹ is nothydrogen.

In further aspects of the invention, pure, in particular, substantiallypure, compounds of the formula III are employed wherein R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are independentlyhydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino, imino, azido,thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo, silyl,silyloxy, silylalkyl, silylthio, ═O, ═S, carboxyl, carbonyl, carbamoyl,or carboxamide, and R¹¹ is alkyl, alkenyl, alkynyl, alkylene,alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy,arylalkoxy, aroyl, heteroaryl, acyl, acyloxy, amino, imino, azido,thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo, silyl,silyloxy, silylalkyl, silylthio, ═O, ═S, carboxyl, carbonyl, carbamoyl,or carboxamide; or an isomer, a pharmaceutically acceptable salt, orderivative thereof. In aspects of the invention R¹¹ is C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy, C₃-C₁₀cycloalkyl, C₄-C₁₀cycloalkenyl, C₃-C₁₀cycloalkoxy, C₆-C₁₀aryl,C₆-C₁₀aryloxy, C₆-C₁₀aryl-C₁-C₃alkoxy, C₆-C₁₀aroyl, C₆-C₁₀heteroaryl,C₃-C₁₀heterocyclic, C₁-C₆acyl, C₁-C₆acyloxy, —NH₂, —NHR²⁸—NR²⁸R²⁹,═NR²⁸, —S(O)₂R²⁸, —SH, —SO₃H, nitro, cyano, halo, haloalkyl, haloalkoxy,hydroxyalkyl, —CO₂H, —CO₂R²⁸, —NHC(O)R²⁸, —C(O)NH₂, —C(O)NHR²⁸,—C(O)NR²⁸R²⁹, —NHS(O)₂R²⁸, wherein R²⁸ and R²⁹ are independentlyselected from C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₁₀cycloalkyl,C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀ aryl C₁-C₃alkyl, C₆-C₁₀heteroaryl and C₃-C₁₀heterocyclic.

In certain aspects a compound of the formula III is employed wherein R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are hydrogen,hydroxyl, alkyl, and one or both of R¹⁰ and R¹¹ are independentlysubstituted or unsubstituted hydrogen, hydroxyl, alkyl, alkenyl,alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl,cycloalkenyl, aryl, aryloxy, arylalkoxy, aroyl, heteroaryl,heterocyclic, acyl, acyloxy, sulfonyl, sulfinyl, sulfenyl, amino, imino,azido, thiol, thioalkyl, thioalkoxy, thioaryl, nitro, ureido, cyano,halo, silyl, silylalkyl, silyloxy, silylthio, ═O, ═S, carboxyl,carbonyl, or carbamoyl, or an isomer or a pharmaceutically acceptablesalt thereof. In aspects of the invention one or both of R¹⁰ and R¹¹ areindependently C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxy,C₂-C₆ alkenyloxy, C₃-C₁₀ cycloalkyl, C₄-C₁₀cycloalkenyl,C₃-C₁₀cycloalkoxy, C₆-C₁₀aryl, C₆-C₁₀aryloxy, C₆-C₁₀aryl-C₁-C₃alkoxy,C₆-C₁₀aroyl, C₆-C₁₀heteroaryl, C₃-C₁₀heterocyclic, C₁-C₆acyl,C₁-C₆acyloxy, —NH₂, —NHR²⁸, —NR²⁸R²⁹, ═NR²⁸, —S(O)₂R²⁸, —SH, —SO₃H,nitro, cyano, halo, haloalkyl, haloalkoxy, hydroxyalkyl, —CO₂H, —CO₂R²⁸,—NHC(O)R²⁸, —C(O)NH₂, —C(O)NHR²⁸, —C NR²⁸R²⁹, —NHS(O)₂R²⁸, wherein R²⁸and R²⁹ are independently selected from C₁-C₆alkyl, C₂-C₆alkenyl,C₂-C₆alkynyl, C₃-C₁₀cycloalkyl, C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl,C₆-C₁₀aryl C₁-C₃alkyl, C₆-C₁₀ heteroaryl and C₃-C₁₀heterocyclic.

In certain aspects a compound of the formula III is employed wherein R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are hydrogen; andR¹⁰ is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxy, C₂-C₆alkenyloxy, C₃-C₁₀ cycloalkyl, C₄-C₁₀cycloalkenyl, C₃-C₁₀cycloalkoxy,C₆-C₁₀aryl, C₆-C₁₀aryloxy, C₆-C₁₀aryl-C₁-C₃alkoxy, C₆-C₁₀aroyl,C₆-C₁₀heteroaryl, C₃-C₁₀heterocyclic, C₁-C₆acyl, C₁-C₆acyloxy, —NH₂,—NHR²⁸, —NR²⁸R²⁹, ═NR²⁸—S(O)₂R²⁸, —SH, —SO₃H, nitro, cyano, halo,haloalkyl, haloalkoxy, hydroxyalkyl, —CO₂H, —CO₂R²⁸, —NHC(O)R²⁸,—C(O)NH₂, —C(O)NHR²⁸, —C(O)NR²⁸R²⁹, —NHS(O)₂R²⁸ wherein R²⁸ and R²⁹ areindependently selected from C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₁₀cycloalkyl, C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀ arylC₁-C₃alkyl, C₆-C₁₀ heteroaryl and C₃-C₁₀heterocyclic.

In certain aspects a compound of the formula III is employed wherein R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are hydrogen; R¹⁰is hydrogen, hydroxyl, alkyl (e.g., C₁-C₆ alkyl), aryl [e.g.,C₆-C₁₀aryl, in particular, phenyl which is optionally substituted (e.g.,with halide)], C₃-C₁₀heterocyclic (e.g., piperazinyl which may besubstituted, for example substituted with a pyrimidinyl; or morpholinylwhich may be substituted), —NR³OR³¹ wherein R³⁰ is hydrogen or alkyl,and R³¹ is phenyl which may be substituted or alkyl (e.g., C₁-C₆ alkyl)which may be substituted [e.g. with amino, in particular —CH₂CH₂NH₂;CH₂CH₂NHCOOC(CH₃)₃], or —SR³² wherein R³² is phenyl which may besubstituted; and R¹¹ is hydrogen, alkyl, or aryl (e.g., C6-C₁₀aryl, inparticular, e.g. phenyl) which may be substituted.

In aspects of the invention R¹¹ is alkyl, halo, aryl, substituted aryl(e.g. alkylaryl), or an unsaturated 5 to 6 membered heteromonocyclylgroup containing 1 to 4 nitrogen atoms In an embodiment R¹¹ is loweralkyl (e.g., C₁-C₆ alkyl) or a branched alkyl. In another embodiment,R¹¹ is C₆-C₁₀aryl, in particular phenyl. In another embodiment, R¹¹ ishalo. In a still further embodiment, R¹¹ is an unsaturated 5 to 6membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, forexample, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl,3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazolyl, tetrazolyl and the like. In a particular embodiment, R¹¹ ispyridinyl.

In certain aspects of the invention a compound of the formula III isemployed wherein R¹⁰ is hydrogen, halo, optionally substituted hydroxyl,alkyl, pyridinyl, phenyl, benzyl, piperazinyl, amino, morpholinyl, or—SR³³ wherein R³³ is alkyl or aryl. In an embodiment, R¹⁰ is—NH[CH₂]_(m)NR³⁴R³⁵ wherein m is 1 to 6, in particular 2 to 4, R³⁴ ishydrogen, R³⁵ is a carboxyl, in particular —COOC(CH₃)₃.

In particular embodiments of the invention, one of R¹⁰ and R¹¹ in acompound of the formula III is a heteroaryl in particular an unsaturated5 to 6 membered heteromonocyclyl group containing 1 to 4 nitrogen atoms,more particularly pyridinyl, and the other of R¹⁰ and R¹¹ is hydrogen.

In an aspect of the invention a compound of the formula III is employedwherein R¹¹ is hydrogen, halo, optionally substituted alkyl, pyridinyl,piperidinyl, morpholinyl, piperazinyl, or phenyl.

In aspects of the invention, a compound of the formula III is usedwherein R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷are hydrogen, alkyl, alkoxy, sulfonyl, sulfinyl, halo, thiol, orcarboxyl, and R¹¹ is alkyl, alkenyl, alkoxy, alkenyloxy, aryl,heteroaryl, acyl, acyloxy, amino, imino, azido, thiol, thioalkyl,thioalkoxy, thioaryl, nitro, cyano, halo, silyl, ═O, ═S, carboxyl,carbonyl, carbamoyl, or carboxamide; or an isomer or a pharmaceuticallyacceptable salt thereof. In particular aspects, R¹¹ is C₁-C₆ alkyl,C₂-C₆ alkenyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy, C₆-C₁₀aryl,C₆-C₁₀heteroaryl, C₁-C₆acyl, C₁-C₆acyloxy, —NH₂, —NHR²⁸, —NR²⁸R²⁹,═NR²⁸, —S(O)₂R²⁸, —SH, —SO₃H, nitro, cyano, halo, haloalkyl, haloalkoxy,—CO₂H, —CO₂R²⁸, —NHC(O)R²⁸, —C(O)NH₂, —C(O)NHR²⁸, —C(O)NR²⁸R²⁹,—NHS(O)₂R²⁸, wherein R²⁸ and R²⁹ are independently selected fromC₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₁₀cycloalkyl,C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀ aryl C₁-C₃alkyl, C₆-C₁₀heteroaryl and C₃-C₁₀heterocyclic.

In aspects of the invention, a compound of the formula III is employedwherein R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷are hydrogen, and R¹¹ is alkyl, alkenyl, alkynyl, alkylene, alkoxy,aryl, or an unsaturated 5 to 6 membered heteromonocyclyl groupcontaining 1 to 4 nitrogen atoms. In particular aspects, R⁴, R⁵, R⁶, R⁷,R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are hydrogen and R¹¹ isalkyl or pyridinyl, more particularly R¹¹ is alkyl.

In other aspects of the invention, one of R¹⁰ and R¹¹ in a compound ofthe formula III is alkyl, in particular C₁-C₆ alkyl and the other of R¹⁰and R¹¹ is hydrogen.

In particular embodiments of the invention, one of R¹⁰ and R¹¹ in acompound of the formula III is aryl in particular C₆-C₁₀aryl, moreparticularly phenyl or benzyl, and the other of R¹⁰ and R¹¹ is hydrogen.

In embodiments of the invention, the compound of the formula II is acompound in Table 1 or 2.

In particular embodiments of the invention, the compound of the formulaIII is MW01-6-189WH, MW01-5-188WH, or MW01-2-151SRM, and/or a salt orderivatives thereof.

In more particular embodiments, the compound of the formula II is4-methyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-2-151SRM), and/or a salt or derivative thereof.

In more particular embodiments, the compound of the formula II is4,6-diphenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-5-188WH), and/or a salt or derivative thereof.

A compound of the formula I, II or III may be in the form of a prodrugthat is converted in vivo to an active compound. For example, in acompound of the formula II one or more of R¹⁰ and R¹¹ may comprise acleavable group that is cleaved after administration to a subject toprovide an active (e.g., therapeutically active) compound, or anintermediate compound that subsequently yields the active compound. Acleavable group can be an ester that is removed either enzymatically ornon-enzymatically.

A compound of the formula I, II or III may comprise a carrier, such asone or more of a polymer, carbohydrate, peptide or derivative thereof,which may be directly or indirectly covalently attached to the compound.A carrier may be substituted with substituents described hereinincluding without limitation one or more alkyl, amino, nitro, halogen,thiol, thioalkyl, sulfate, sulfonyl, sulfinyl, sulfoxide, hydroxylgroups. In aspects of the invention the carrier is an amino acidincluding alanine, glycine, praline, methionine, serine, threonine,asparagine, alanyl-alanyl, prolyl-methionyl, or glycyl-glycyl. A carriercan also include a molecule that targets a compound of the formula I, IIor III to a particular tissue or organ. Thus, a carrier may facilitateor enhance transport of a compound of the formula I, II or III to thebrain.

Compounds of the formula I, II or III can be prepared using reactionsand methods generally known to the person of ordinary skill in the art,having regard to that knowledge and the disclosure of this applicationincluding the Examples. The reactions are performed in a solventappropriate to the reagents and materials used and suitable for thereactions being effected. It will be understood by those skilled in theart of organic synthesis that the functionality present on the compoundsshould be consistent with the proposed reaction steps. This willsometimes require modification of the order of the synthetic steps orselection of one particular process scheme over another in order toobtain a desired compound of the invention. It will also be recognizedthat another major consideration in the development of a synthetic routeis the selection of the protecting group used for protection of thereactive functional groups present in the compounds described in thisinvention. An authoritative account describing the many alternatives tothe skilled artisan is Greene and Wuts (Protective Groups In OrganicSynthesis, Wiley and Sons, 1991).

The starting materials and reagents used in preparing compounds or theinvention are either available from commercial suppliers or are preparedby methods well known to a person of ordinary skill in the art,following procedures described in such references as Fieser and Fieser'sReagents for Organic Synthesis, vols. 1-17, John Wiley and Sons, NewYork, N.Y., 1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 andsupps., Elsevier Science Publishers, 1989; Organic Reactions, vols.1-40, John Wiley and Sons, New York, N.Y., 1991; March J.: AdvancedOrganic Chemistry, 4th ed., John Wiley and Sons, New York, N.Y.; andLarock: Comprehensive Organic Transformations, VCH Publishers, New York,1989.

The starting materials, intermediates, and compounds of the formula I,II or III may be isolated and purified using conventional techniques,such as precipitation, filtration, distillation, crystallization,chromatography, and the like. The compounds of the formula I, II or IIImay be characterized using conventional methods, including physicalconstants and spectroscopic methods, in particular HPLC.

The compounds of the formula I, II or III which are basic in nature canform a wide variety of different salts with various inorganic andorganic acids. In practice is it desirable to first isolate a compoundof the formula I, II or III from the reaction mixture as apharmaceutically unacceptable salt and then convert the latter to thefree base compound by treatment with an alkaline reagent andsubsequently convert the free base to a pharmaceutically acceptable acidaddition salt. The acid addition salts of the base compounds of theformula I, II or III are readily prepared by treating the base compoundwith a substantially equivalent amount of the chosen mineral orinorganic or organic acid in an aqueous solvent medium or in a suitableorganic solvent such as methanol or ethanol. Upon careful evaporation ofthe solvent, the desired solid salt is obtained.

Compounds of the formula I, II or III which are acidic in nature arecapable of forming base salts with various pharmacologically acceptablecations. These salts may be prepared by conventional techniques bytreating the corresponding acidic compounds with an aqueous solutioncontaining the desired pharmacologically acceptable cations and thenevaporating the resulting solution to dryness, preferably under reducedpressure. Alternatively, they may be prepared by mixing lower alkanolicsolutions of the acidic compounds and the desired alkali metal alkoxidetogether and then evaporating the resulting solution to dryness in thesame manner as before. In either case, stoichiometric quantities ofreagents are typically employed to ensure completeness of reaction andmaximum product yields.

In particular aspects, the present invention provides methods of makingthe compounds disclosed herein, comprising the steps provided (See, forexample, the Figures and Examples).

In an aspect, the invention provides a process for preparing a compoundof the formula III wherein R¹¹ is hydrogen and R¹⁰ is an unsaturated 5to 6 membered heteromonocyclyl group containing 1 to 4 nitrogen atoms,in particular, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl,3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazolyl, or tetrazolyl, more particularly pyridinyl, which comprisesreacting a compound of the formula III wherein R¹⁰ is halo, inparticular chloro, and R¹¹ is hydrogen with boronic acid substitutedwith an unsaturated 5 to 6 membered heteromonocyclyl group containing 1to 4 nitrogen atoms, in particular, pyrrolyl, pyrrolinyl, imidazolyl,pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl,pyrazinyl, pyridazinyl, triazolyl, or tetrazolyl, more particularlypyridinyl, under suitable conditions to prepare a compound of theformula III wherein R¹¹ is hydrogen and R¹⁰ is an unsaturated 5 to 6membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, inparticular, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl,3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazolyl, or tetrazolyl, more particularly pyridinyl.

In another aspect, the invention provides a process for preparing acompound of the formula III wherein R¹¹ is hydrogen and R¹⁰ is asubstituted aryl which comprises reacting a compound of the formula IIIwherein R¹⁰ is halo, in particular chloro, and R¹¹ is hydrogen, with asubstituted aryl boronic acid under suitable conditions to prepare acompound of the formula III wherein R¹¹ is hydrogen and R¹⁰ is asubstituted aryl.

In another aspect, the invention provides a process for preparing acompound of the formula III wherein R¹⁰ is hydrogen and R¹¹ is alkylwhich comprises reacting a compound of the formula III wherein R¹¹ ishalo, in particular chloro, and R¹⁰ is hydrogen, with an alkyl boronicacid under suitable conditions to prepare a compound of the formula IIIwherein R¹⁰ is hydrogen and R¹¹ is alkyl. In an embodiment, R¹¹ is loweralkyl, in particular methyl or ethyl, and a compound of the formula IIIwherein R¹¹ is chloro is reacted with lower alkyl boronic acid, inparticular methyl or ethyl boronic acid under suitable conditions.

In another aspect, the invention provides a process for preparing acompound of the formula III wherein R¹⁰ is hydrogen and R¹¹ is arylwhich comprises reacting a compound of the formula III wherein R¹⁰ ishydrogen and R¹¹ is halo (e.g., chloro), with pyridazine substituted atthe C3 position with halo (e.g., chloro), at the C4 position with aryl,and at the 6 position with phenyl, with 2-(piperidin-4-yloxy)pyrimidineunder suitable conditions to prepare a compound of the formula IIIwherein R¹⁰ is hydrogen and R¹¹ is aryl. In an embodiment, R¹¹ isphenyl.

In another aspect, the invention provides a process for preparing acompound of the formula III wherein R¹⁰ is hydrogen and R¹¹ is anunsaturated 5 to 6 membered heteromonocyclyl group containing 1 to 4nitrogen atoms, in particular, pyrrolyl, pyrrolinyl, imidazolyl,pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl,pyrazinyl, pyridazinyl, triazolyl, or tetrazolyl, more particularlypyridinyl which comprises reacting a compound of the formula III whereinR¹¹ is halo, in particular chloro, and R¹⁰ is hydrogen, with a boronicacid substituted with an unsaturated 5 to 6 membered heteromonocyclylgroup containing 1 to 4 nitrogen atoms, in particular, pyrrolyl,pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, ortetrazolyl, more particularly pyridinyl, under suitable conditions toprepare a compound of the formula III wherein R¹⁰ is hydrogen and R¹¹ isan unsaturated 5 to 6 membered heteromonocyclyl group containing 1 to 4nitrogen atoms, in particular, pyrrolyl, pyrrolinyl, imidazolyl,pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl,pyrazinyl, pyridazinyl, triazolyl, or tetrazolyl, more particularlypyridinyl.

In an embodiment, the invention provides a process for preparing acompound of the formula III wherein R¹⁰ is hydrogen and R¹¹ is pyridinylwhich comprises reacting a compound of the formula III wherein R¹¹ ishalo, in particular chloro, and R¹⁰ is hydrogen, with a pyridinylboronic acid under suitable conditions to prepare a compound of theformula III wherein R¹⁰ is hydrogen and R¹¹ is pyridinyl.

In another aspect, the invention provides a process for preparing acompound of the formula III wherein R¹⁰ is hydrogen and R¹¹ is anunsaturated 5 to 6 membered heteromonocyclyl group containing 1 to 4nitrogen atoms, in particular, pyrrolyl, pyrrolinyl, imidazolyl,pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl,pyrazinyl, pyridazinyl, triazolyl, or tetrazolyl, more particularlypyridinyl which comprises reacting a pyridazine substituted at the C3position with halo, at the C4 position with an unsaturated 5 to 6membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, inparticular, pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl,3-pyridyl, 4-pyridyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl,triazolyl, or tetrazolyl, more particularly pyridinyl, and at the 6position with phenyl, with 2-(piperidin-4-yloxy)pyrimidine undersuitable conditions to prepare a compound of the formula III wherein R¹⁰is hydrogen and R¹¹ is an unsaturated 5 to 6 membered heteromonocyclylgroup containing 1 to 4 nitrogen atoms, in particular, pyrrolyl,pyrrolinyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl,pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazolyl, ortetrazolyl, more particularly pyridinyl.

In an embodiment, the invention provides a process for preparing acompound of the formula III wherein R¹⁰ is hydrogen and R¹¹ is pyridinylwhich comprises reacting a pyridazine substituted at the C3 positionwith halo, at the C4 position with pyridinyl, and at the 6 position withphenyl, with 2-(piperidin-4-yloxy)pyrimidine under suitable conditionsto prepare a compound of the formula III wherein R¹⁰ is hydrogen and R¹¹is pyridinyl.

In another aspect, the invention provides a process for preparing acompound of the formula III wherein R¹⁰ is hydrogen and R¹¹ ispiperidinyl or substituted piperidinyl which comprises reacting acompound of the formula II wherein R¹¹ is halo, in particular chloro,and R¹⁰ is hydrogen with piperazinyl or substituted piperazinyl undersuitable conditions to prepare a compound of the formula II wherein R¹⁰is hydrogen and R¹¹ is piperidinyl or substituted piperidinyl.

In another aspect, the invention provides a process for preparing acompound of the formula III wherein R¹⁰ is hydrogen and R¹¹ is an alkylwhich comprises reacting a pyridazine substituted at the C3 positionwith halo (e.g., chloro), at the C4 position with alkyl, and at the 6position with phenyl, with 2-(piperidin-4-yloxy)pyrimidine undersuitable conditions to prepare a compound of the formula III wherein R¹⁰is hydrogen and R¹¹ is an alkyl. In an embodiment, R¹¹ is methyl orethyl.

In a particular aspect, the invention provides a process for preparing acompound of the formula III wherein R¹⁰ is hydrogen and R¹¹ is alkylcomprising reacting a compound of the formula IV

wherein R^(1′) is halo, in particular chloro or bromo, more particularlychloro and R^(2′) is alkyl with 2-(piperazin-1-yl)pyrimidine undersuitable conditions, in particular under reflux conditions to produce acompound of the formula III wherein R¹⁰ is hydrogen and R¹¹ is alkyl.

Therapeutic efficacy and toxicity of compounds, compositions and methodsof the invention may be determined by standard pharmaceutical proceduresin cell cultures or with experimental animals such as by calculating astatistical parameter such as the ED₅₀ (the dose that is therapeuticallyeffective in 50% of the population) or LD₅₀ (the dose lethal to 50% ofthe population) statistics. The therapeutic index is the dose ratio oftherapeutic to toxic effects and it can be expressed as the ED₅₀/LD₅₀ratio. Pharmaceutical compositions which exhibit large therapeuticindices are preferred. By way of example, one or more of the therapeuticeffects can be demonstrated in a subject or disease model, for example,a TgCRND8 mouse with symptoms of Alzheimer's disease.

Biological investigations were done with compounds disclosed herein thatwere >95% homogenous as determined by HPLC/MS analysis. As part of ahierarchal, cell-based screening protocol, the compounds were screenedfor their ability to block IL-1β and TNFα production by BV-2 mousemicroglial cells stimulated with LPS.

Compositions, Methods and Kits

The invention provides dosage forms, formulations, and methods thatprovide advantages, in particular lower risk of side effects (e.g. lowerrisk of QT-related side effects) and/or beneficial pharmacokineticprofiles, more particularly sustained pharmacokinetic profiles. Acompound of the formula I, II or III can be utilized in dosage forms inpure or substantially pure form, in the form of its pharmaceuticallyacceptable salts, and also in other forms including anhydrous orhydrated forms.

A beneficial pharmacokinetic profile may be obtained by administering aformulation or dosage form suitable for once, twice a day, or threetimes a day or more administration comprising one or more compound ofthe formula I, II or III present in an amount sufficient to provide therequired concentration or dose of the compound to an environment of useto treat a disease disclosed herein, in particular a neuroinflammatorydisease. In an aspect, the environment of use is the brain and/orplasma.

Embodiments of the invention relate to a dosage form comprising one ormore compound of the formula I, II or III that provides peak plasmaconcentrations of the compound, C_(max), of between about 0.001 to 2mg/ml, 0.001 to 1 mg/ml, 0.002 to 2 mg/ml, 0.005 to 2 mg/ml, 0.01 to 2mg/ml, 0.05 to 2 mg/ml, 0.1 to 2 mg/ml, 0.001 to 0.5 mg/ml, 0.002 to 1mg/ml, 0.005 to 1 mg/ml, 0.01 to 1 mg/ml, 0.05 to 1 mg/ml, or 0.1 to 1mg/ml.

In further aspects, the invention provides a formulation or dosage formcomprising one or more compound of the formula I, II or III thatprovides an elimination t_(1/2) of 0.5 to 20 hours, 0.5 to 15 hours, 0.5to 10 hours, 0.5 to 6 hours, 1 to 20 hours, 1 to 15 hours, 1 to 10hours, or 1 to 6 hours.

Further aspects of the invention relate to a formulation or dosage formcomprising one or more compound of the formula I, II or III thatprovides an AUC for plasma of about 3 to 2000 ng·h/ml, 3 to 3000ng·h/ml, 3 to 4000 ng·h/ml, 2 to 2000 ng·h/ml, 2 to 3000 ng·h/ml, 2 to4000 ng·h/ml, 1 to 2000 ng·h/ml, 1 to 3000 ng·h/ml, 1 to 4000 ng·h/ml,1, and in particular 3 to 3000 ng·h/ml

A subject may be treated with a compound of the formula I, II or III orcomposition or unit dosage thereof on substantially any desiredschedule. They may be administered one or more times per day, inparticular 1 or 2 times per day, once per week, once a month orcontinuously. However, a subject may be treated less frequently, such asevery other day or once a week, or more frequently. A compound orcomposition may be administered to a subject for about or at least about24 hours, 2 days, 3 days, 1 week, 2 weeks to 4 weeks, 2 weeks to 6weeks, 2 weeks to 8 weeks, 2 weeks to 10 weeks, 2 weeks to 12 weeks, 2weeks to 14 weeks, 2 weeks to 16 weeks, 2 weeks to 6 months, 2 weeks to12 months, 2 weeks to 18 months, 2 weeks to 24 months, or for more than24 months, periodically or continuously.

In an aspect, a beneficial pharmacokinetic profile can be obtained bythe administration of a formulation or dosage form suitable for once,twice, or three times a day administration, preferably twice a dayadministration comprising one or more compound of the formula I, II orIII present in an amount sufficient to provide the required dose of thecompound. In an aspect, the required dose of a compound of the formulaI, II or III administered once twice, three times or more daily is about0.01 to 3000 mg/kg, 0.01 to 2000 mg/kg, 0.5 to 2000 mg/kg, about 0.5 to1000 mg/kg, 0.1 to 1000 mg/kg, 0.1 to 500 mg/kg, 0.1 to 400 mg/kg, 0.1to 300 mg/kg, 0.1 to 200 mg/kg, 0.1 to 100 mg/kg, 0.1 to 50 mg/kg, 0.1to 20 mg/kg, 0.1 to 10 mg/kg, 0.1 to 6 mg/kg, 0.1 to 5 mg/kg, 0.1 to 3mg/kg, 0.1 to 2 mg/kg, 0.1 to 1 mg/kg, 1 to 1000 mg/kg, 1 to 500 mg/kg,1 to 400 mg/kg, 1 to 300 mg/kg, 1 to 200 mg/kg, 1 to 100 mg/kg, 1 to 50mg/kg, 1 to 20 mg/kg, 1 to 10 mg/kg, 1 to 6 mg/kg, 1 to 5 mg/kg, or 1 to3 mg/kg, or 1 to 2.5 mg/kg, or less than or about 10 mg/kg, 5 mg/kg, 2.5mg/kg, 1 mg/kg, or 0.5 mg/kg twice daily or less

Certain dosage forms and formulations may minimize the variation betweenpeak and trough plasma and/or brain levels of compounds of the formulaI, II or III and in particular provide a sustained therapeuticallyeffective amount of the compounds.

The invention also contemplates a formulation or dosage form comprisingamounts of one or more compound of the formula I, II or III that resultsin therapeutically effective amounts of the compound over a dosingperiod, in particular a 24 hour dosing period. In aspects of theinvention the therapeutically effective amounts of a compound of theformula I, II or III are between about 0.1 to 1000 mg/kg, 0.1 to 500mg/kg, 0.1 to 400 mg/kg, 0.1 to 300 mg/kg, 0.1 to 200 mg/kg, 0.1 to 100mg/kg, 0.1 to 75 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 20mg/kg, 0.1 to 15 mg/kg, 0.1 to 10 mg/kg, 0.1 to 9 mg/kg, 0.1 to 8 mg/kg,0.1 to 7 mg/kg, 0.1 to 6 mg/kg, 0.1 to 5 mg/kg, 0.1 to 4 mg/kg, 0.1 to 3mg/kg, 0.1 to 2 mg/kg, or 0.1 to 1 mg/kg.

A medicament or treatment of the invention may comprise a unit dosage ofat least one compound of the formula I, II or III to provide therapeuticeffects. A “unit dosage” or “dosage unit” refers to a unitary, i.e. asingle dose, which is capable of being administered to a patient, andwhich may be readily handled and packed, remaining as a physically andchemically stable unit dose comprising either the active agents as suchor a mixture with one or more solid or liquid pharmaceutical excipients,carriers, or vehicles.

A formulation or dosage form of the invention may be an immediaterelease dosage form or a non-immediate release delivery system,including without limitation a delayed-release or sustained-releasedosage form.

In aspects, the invention provides a sustained-release dosage form of acompound of the formula I, II or III which advantageously achieves amore sustained drug plasma and/or brain level response while mitigatingor eliminating drug concentration spikes by providing a substantiallysteady release of the compound over time. A substantially constantplasma concentration preferably correlates with one or more therapeuticeffects disclosed herein. In embodiments, the sustained-release dosageform is for oral administration.

A composition, in particular a dosage form or formulation, may be in anyform suitable for administration to a subject, including withoutlimitation, a form suitable for oral, parenteral, intravenous (bolus orinfusion), intraperitoneal, subcutaneous, or intramuscularadministration. A dosage form or formulation may be a pill, tablet,caplet, soft and hard gelatin capsule, lozenge, sachet, cachet, vegicap,liquid drop, elixir, suspension, emulsion, solution, syrup, aerosol (asa solid or in a liquid medium) suppository, sterile injectable solution,and/or sterile packaged powder.

In an aspect of the invention a dosage form or formulation is an oraldosage form or formulation such as tablets, caplets, soft and hardgelatin capsules, pills, powders, granules, elixirs, tinctures,suspensions, syrups, and emulsions. In another aspect the dosage form orformulation is a parenteral dosage form such as an active substance in asterile aqueous or non-aqueous solvent, such as water, isotonic saline,isotonic glucose solution, buffer solution, or other solventsconveniently used for parenteral administration.

A compound of the formula I, II or III of the invention may beformulated into a pharmaceutical composition for administration to asubject by appropriate methods known in the art. Pharmaceuticalcompositions of the present invention or fractions thereof comprisesuitable pharmaceutically acceptable carriers, excipients, and vehiclesselected based on the intended form of administration, and consistentwith conventional pharmaceutical practices. Suitable pharmaceuticalcarriers, excipients, and vehicles are described in the standard text,Remington: The Science and Practice of Pharmacy (21^(st) Edition. 2005,University of the Sciences in Philadelphia (Editor), Mack PublishingCompany), and in The United States Pharmacopeia: The National Formulary(USP 24 NF19) published in 1999. By way of example for oraladministration in the form of a capsule or tablet, the active componentscan be combined with an oral, non-toxic pharmaceutically acceptableinert carrier such as lactose, starch, sucrose, methyl cellulose,magnesium stearate, glucose, calcium sulfate, dicalcium phosphate,mannitol, sorbital, and the like. For oral administration in a liquidform, the drug components may be combined with any oral, non-toxic,pharmaceutically acceptable inert carrier such as ethanol, glycerol,water, and the like. Suitable binders (e.g. gelatin, starch, cornsweeteners, natural sugars including glucose; natural and syntheticgums, and waxes), lubricants (e.g. sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, and sodiumchloride), disintegrating agents (e.g. starch, methyl cellulose, agar,bentonite, and xanthan gum), flavoring agents, and coloring agents mayalso be combined in the compositions or components thereof. Compositionsas described herein can further comprise wetting or emulsifying agents,or pH buffering agents.

A composition of the invention can be a liquid solution, suspension,emulsion, tablet, pill, capsule, sustained release formulation, orpowder. The compositions can be formulated as a suppository, withtraditional binders and carriers such as triglycerides. Oralformulations can include standard carriers such as pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, etc. Various delivery systems are knownand can be used to administer a composition of the invention, e.g.encapsulation in liposomes, microparticles, microcapsules, and the like.

Formulations for parenteral administration may include aqueoussolutions, syrups, aqueous or oil suspensions and emulsions with edibleoil such as cottonseed oil, coconut oil or peanut oil. Dispersing orsuspending agents that can be used for aqueous suspensions includesynthetic or natural gums, such as tragacanth, alginate, acacia,dextran, sodium carboxymethylcellulose, gelatin, methylcellulose, andpolyvinylpyrrolidone.

Compositions for parenteral administration may include sterile aqueousor non-aqueous solvents, such as water, isotonic saline, isotonicglucose solution, buffer solution, or other solvents conveniently usedfor parenteral administration of therapeutically active agents. Acomposition intended for parenteral administration may also includeconventional additives such as stabilizers, buffers, or preservatives,e.g. antioxidants such as methylhydroxybenzoate or similar additives.

A composition of the invention may be sterilized by, for example,filtration through a bacteria retaining filter, addition of sterilizingagents to the composition, irradiation of the composition, or heatingthe composition. Alternatively, the compounds or compositions of thepresent invention may be provided as sterile solid preparations e.g.lyophilized powder, which are readily dissolved in sterile solventimmediately prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of a composition of the invention, suchlabeling would include amount, frequency, and method of administration.

According to the invention, a kit is provided. In an aspect, the kitcomprises a compound of the formula I, II or III or a formulation of theinvention in kit form. The kit can be a package which houses a containerwhich contains compounds of the formula I, II or III or formulations ofthe invention and also houses instructions for administering thecompounds or formulations to a subject. The invention further relates toa commercial package comprising compounds of the formula I, II or III orformulations of the invention together with instructions forsimultaneous, separate or sequential use. In particular a label mayinclude amount, frequency, and method of administration.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of acomposition of the invention to provide a therapeutic effect. Associatedwith such container(s) can be various written materials such asinstructions for use, or a notice in the form prescribed by agovernmental agency regulating the labeling, manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use, or sale for human administration.

The invention also relates to articles of manufacture and kitscontaining materials useful for treating a disease disclosed herein. Anarticle of manufacture may comprise a container with a label. Examplesof suitable containers include bottles, vials, and test tubes which maybe formed from a variety of materials including glass and plastic. Acontainer holds compounds of the formula I, II or III or formulations ofthe invention which are effective for treating a disease disclosedherein. The label on the container indicates that the compounds of theformula I, II or III or formulations of the invention are used fortreating a disease disclosed herein and may also indicate directions foruse. In aspects of the invention, a medicament or formulation in acontainer may comprise any of the medicaments or formulations disclosedherein.

The invention also contemplates kits comprising one or more of compoundsof the formula I, II or III. In aspects of the invention, a kit of theinvention comprises a container described herein. In particular aspects,a kit of the invention comprises a container described herein and asecond container comprising a buffer. A kit may additionally includeother materials desirable from a commercial and user standpoint,including, without limitation, buffers, diluents, filters, needles,syringes, and package inserts with instructions for performing anymethods disclosed herein (e.g., methods for treating a disease disclosedherein). A medicament or formulation in a kit of the invention maycomprise any of the formulations or compositions disclosed herein.

In aspects of the invention, the kits may be useful for any of themethods disclosed herein, including, without limitation treating asubject suffering from Alzheimer's disease. Kits of the invention maycontain instructions for practicing any of the methods described herein.

The compositions and methods described herein are indicated astherapeutic agents or methods either alone or in conjunction with othertherapeutic agents or other forms of treatment. They may beco-administered, combined or formulated with one or more therapies oragents used to treat a condition described herein. Compositions of theinvention may be administered concurrently, separately, or sequentiallywith other therapeutic agents or therapies. Therefore, compounds of theformula I, II or III may be co-administered with one or more additionaltherapeutic agents for treating diseases disclosed herein includingwithout limitation beta-secretase inhibitors, alpha-secretaseinhibitors, and epsilon-secretase inhibitors, acetylcholinesteraseinhibitors, agents that are used for the treatment of complicationsresulting from or associated with a disease disclosed herein, or generalmedications that treat or prevent side effects.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner.

EXAMPLES Example 1 Synthesis of Pyridazine Compounds

The structures of MW01-2-151SRM, MW01-6-189WH, MW01-7-107WH,MW01-4-179LKM, WM01-7-084WH, MH01-7-085WH, MW01-7-133WH, and MW01-7-057are shown in FIG. 1 and synthetic schemes for producing the compoundsare described below.

A. Preparation of 2-(4-(6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine(MW01-3-183WH)

FIG. 2 depicts a synthetic scheme for the preparation of2-(4-(6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine (MW01-3-183WH).Reagents and conditions: (a) 1-BuOH, NH₄Cl, and2-(piperazin-1-yl)pyrimidine. A typical reaction mixture comprisingabout 0.01 mol of 3-chloro-6-phenylpyridazine by2-(piperazin-1-yl)pyrimidine, about 0.05 mol of2-(piperazin-1-yl)pyrimidine and about 0.01 mol of ammoniumhydrochloride was prepared in about 15 ml of 1-BuOH. The mixture wasstirred at about 130° C. for about 48 h, and then the solvent wasremoved under reduced pressure. The remaining residue was then extractedwith ethyl acetate, washed with water and brine, dried over anhydrousNa₂SO₄. Removal of solvent followed by recrystallization from 95%ethanol yielded light yellow crystals, yield 96.4%; HPLC: 97.4% purity;HRMS calculated 318.1587, found 318.1579; 1H NMR (CDCl₃): d 8.356 (d,J=4.5, 2H), 8.011 (d, J=7.5, 11 2H), 7.692 (d, J=9.5, 1H), 7.468 (t,J=6.0, 2H), 7.417 (d, J=7.5, 1H), 7.047 (d, J=9.5, 1H), 6.546 (t, J=4.5,1H), 4.013 (t, J=5.0, 4H), 3.826 (t, J=5.0, 4H).

B. Preparation of4-methyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-2-151SRM)

4-methyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-2-151 SRM) was prepared by several synthetic schemes as depictedin FIG. 3 (Scheme 1), FIG. 4 (Scheme 2), FIG. 5 (Scheme 3), and FIG. 6(Scheme 4), which were carried out as described in detail herein. Thevarious reaction schemes (Schemes 1, 2, and 3) are generally applicableto the compounds of the present invention and are not restricted inutility only to the preparation of MW01-2-151 SRM.

Scheme 1 (FIG. 3) 4,5-dihydro-4-methyl-6-phenylpyridazin-3(2H)-one (2)

A 250 mL three-neck round bottom flask fit with a temperature probe andcondenser is charged with 7.7 g (40 mmole) of2-methyl-4-oxo-4-phenylbutanoic acid 1 and 20 ml of ethanol (95%). Thesuspension is cooled to below 10° C. and 2.2 ml (42 mmole, 1.05 equiv.)of hydrazine monohydrate in 10 mL of ethanol is added dropwise at a ratethat maintains the solution temperature at below 20° C. Upon addition,the suspension changes to a pale yellow solution. After addition, thereaction mixture is heated to reflux and stirred for 2 h, and after 20minutes of heating, a solid is seen in the mixture. Once the reaction iscompleted, the flask is removed from the oil bath and cooled to ambienttemperature. Upon cooling, white crystals form in the flask, which arecollected by filtration. The solid is washed first with 30 mL of 2NNaHCO₃, followed by 60 mL Milli-Q water three times, and dried over amedium frit sintered glass funnel in vacuo to give the desired product 2in 96.1% yield. [See Hansen, K B et al. Organic process research &development, 2005, 9, 634-639; Nelson, D A. US 20050137397A1. Coudert, Pet al. Journal of Heterocyclic Chemistry, 1988, 25(3), 799-802.]

4-methyl-6-phenylpyridazin-3(2H)-one (3)

7.0 g (35 mmole) of 2 is placed in a 250 ml single-necked round bottomflask followed by 30 mL of acetonitrile. The mixture is stirred to allow2 to dissolve. 11.3 g (84 mmole, 2.4 equiv.) of anhydrous copper (II)chloride is added to the solution to give a green-yellow suspension. Areflux condenser is connected to the flask and a dry tube filled withanhydrous CaCl₂ is fitted to the top of the condenser. To control theHCl gas that forms during the course of the reaction, a NaOH solution isused to absorb the HCl that escapes from dry tube. The reaction mixtureis heated to reflux, and the color of the reaction suspension changes todark green upon heating. When the reaction is complete (after refluxingfor 2 h), the flask is removed from the oil bath and cooled to ambienttemperature. The reaction is cooled in an ice-water bath and 150 mL ofice-water is added to quench the reaction. The mixture is stirredvigorously for 10 minutes to give a gray precipitate and blue liquidcontaining copper (I) chloride. The precipitate is collected byfiltration (pH of the filtrate is 0-1) and washed with 100 mL of 1N HClsolution, then 100 mL of water 5 times. To remove remaining copperby-products that are trapped in the solid, the filter cake is stirred in150 mL of 1N HCl solution for 0.5 h and filtered. The filter cake issubsequently washed with Milli-Q water until the filtrate is at pH 7(approximately 7 washes). The solid is dried over a medium frit sinteredglass funnel in vacuo to give 3 as a light gray powder in 93.8% yield.[See Eddy, S et al. Synthetic Communications, 2000, 30(1), 1-7. Csende,F et al. Synthesis, 1995, 1240-1242.]

3-chloro-4-methyl-6-phenylpyridazine (4)

6.0 g (32 mmole) of 3 is placed in a 250 mL single neck round bottomflask and 30 mL of acetonitrile is added to create a pale yellow slurry.6.0 ml (64 mmole, 2 equiv.) of phosphorus oxychloride is added changingthe slurry to a darker color. The flask is fitted with a refluxcondenser and a dry tube filled with anhydrous CaCl₂ is fitted to thetop of the condenser. The reaction mixture is heated at reflux andbecomes a dark red liquid. After the reaction is completed (2.5 h), themixture is cooled to ambient temperature and placed in an ice waterbath. Ice water (150 mL) is slowly poured into the reaction mixture withstirring to decompose the phosphorus oxychloride into IC₁ and H₃PO₄,resulting in formation of a pink solid. The solid is collected byfiltration and washed three times with 50 mL of Milli-Q water. The solidis transferred to a 250 mL beaker, followed by addition of 100 mL ofwater to form a suspension. Subsequently, 1N NaOH is added until theaqueous suspension is at pH=8, and the mixture is stirred for 5 minutesto remove all trace of starting material contaminants. The solid isfiltered and washed 3 times with 100 mL of water to wash out the excessbase. The solid is dried over a medium frit sintered glass funnel invacuo to provide 4 as a light pink powder in 96% yield. [See Contreras,J M et al. Journal of Medicinal Chemistry, 2001, 44(17), 2707-2718;Nelson, D A. US 20050137397A1.]

2-(4-(4-methyl-6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine (5)

7.5 g (36.6 mmole) of 4 is placed in a 250 mL single neck round bottomflask and suspended in 125 mL of water. 60.17 g (366.0 mmole, 10 equiv.)of 2-(piperazine-1-yl)pyrimidine is added and the flask fit with acondenser. The reaction mixture is heated at reflux with rapid stirringfor 60 h, with continuous amine addition possible to boost reactionrates. When complete, the reaction mixture is cooled to ambienttemperature and two layers are observed in the flask consisting of anorange aqueous layer and a brown oil that settles to the bottom of theflask. The water is decanted off, leaving the oil, which is the product5. The oil is then dissolved in minimal volume of isopropanol and heatedto reflux. After 10 minutes of reflux, the solution is cooled to ambienttemperature, and cooled to 0° C. to induce crystallization. Pale yellowcrystals are filtered from isopropanol and rinsed with minimal coldether to provide 5. Recovery of the crystals is 50%, but may beincreased by recursive crystallization of compound. [Contreras, J M etal. Journal of Medicinal Chemistry, 1999, 42(4), 730-741. Chayer, S etal. Tetrahedron Letters, 1998, 39, 841-844.]

Scheme 2 (FIG. 4)

3-chloro-6-phenylpyridazin-4-ol was synthesized according to theprocedure described by Coudert, P., et al., supra.

6-phenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazin-4-ol(MW01-1-7-121 WH)

This compound was prepared from 3-chloro-4-hydroxy-6-phenylpyridazine(14 g, 68 mmol) in the same manner as described below, yielding whitesolid (22.1 g, 66 mmol, 97.3%). ESI-MS: m/z 335.2 (M+H+). 1H NMR (DMSO):1H NMR (DMSO): d 8.406 (d, J=6.5, 2H), 7.740 (d, J=4.0, 2H), 7.558 (s,3H), 6.686 (t, J=4.8, J=4.4, 1H), 6.841 (s, 1H), 3.881 (s, 4H), 3.620(s, 4H), 3.776 (s, 4H).

4-chloro-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-6-127WH)

6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazin-4-ol (22.0 g, 66mmol) was suspended in 75 ml phosphorus oxychloride and heated withstirring at 100° C. for 3 h. After cooling to room temperature themixture was poured onto crushed ice. The mixture was then neutralizedwith NaOH solution to give white suspension. The precipitation wasfiltered off, washed with water, dried over filter funnel to providewhite solid (21.3 g, 60.3 mmol, 91.4%). ESI-MS: m/z 353.4 (M+H+). 1H NMR(CDCl₃): d 8.377 (d, J=4.5, 2H), 8.036 (d, J=7.5, 2H), 7.833 (s, 1H),7.508 (m, 3H), 6.564 (t, J=4.5, 1H), 4.073 (t, J=4.0, J=4.5, 4H), 3.672(t, J=4.0, J=4.5, 4H).

4-methyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-2-151 SRM)

Into a reaction tube were added MW01-6-127WH (1.4 g, 4.0 mmol), K₂CO₃powder (1.7 g, 12.4 mmol), Pd(dppf)Cl₂ (326 mg, 0.4 mmol), silver oxide(2.3 g, 10 mmol), methylboronic acid (324 mg, 5.4 mmol) and 20 ml ofTHF. Argon was then flushed through the tube for 3 min. The tube wasthen sealed tightly and heated with stirring at 80 degree for 12 h.After cooled down, the mixture was quenched with 10% NaOH solution andextracted with ethyl acetate. The organic phase was concentrated invacuo and the residue was purified by column chromatography eluting with1:4, Ethyl Acetate:Petroleum ether. White powder solid was obtained(0.60 g, 1.8 mmol, yield 45.2%). ESI-MS: m/z 333.4 (M+H+). 1H NMR(CDCl₃): d 8.380 (d, J=5.0, 2H), 7.065 (d, J=7.0, 2H), 7.626 (s, 1H),7.473 (m, 3H), 6.567 (t, J=4.5, J=5.0, 1H), 4.056 (t, J=5.0, 4H), 3.475(t, J=5.0, 4H), 2.456 (s, 3H).

Scheme 3 (FIG. 5)

Into a reaction tube were added MW01-6-127WH (1.4 g, 4.0 mmol), K₂CO₃powder (1.7 g, 12.4 mmol), Pd(PPh₃)₄ (240 mg, 0.2 mmol), silver oxide(2.3 g, 10 mmol), methylboronic acid (324 mg, 5.4 mmol) and 20 ml ofDME. Argon was then flushed through the tube for 3 min. The tube wasthen sealed tightly and heated with stirring at 120° C. for 24 h. Aftercooled down, the mixture was filter through acelite earth, the filtratewas then concentrated and the residue was purified by columnchromatography eluting with 1:4, Ethyl Acetate:Petroleum ether. Whitepowder solid was obtained (0.64 g, 1.93 mmol, yield 48.1%). ESI-MS: m/z333.4 (M+H+). 1H NMR (CDCl₃): d 8.380 (d, J=5.0, 2H), 7.065 (d, J=7.0,2H), 7.626 (s, 1H), 7.473 (m, 3H), 6.567 (t, J=4.5, J=5.0, 1H), 4.056(t, J=5.0, 4H), 3.475 (t, J=5.0, 4H), 2.456 (s, 3H).

Scheme 4 (FIG. 6) 4,5-dihydro-4-methyl-6-phenylpyridazin-3(2H)-one(MW01-8-004WH)

7.7 g (40 mmole) of 2-methyl-4-oxo-4-phenylbutanoic acid was added to a100 ml single-necked round bottom flask followed by 3.0 ml (60 mmole) ofhydrazine monohydrate and then 20 ml of reagent grade ethanol (100%, 95%of ethanol should be fine also). The flask was fitted with a refluxcondenser and the reaction mixture was heated to reflux in an oil bathat 110° C. (temperature of oil bath) and stirred for 2 h. The flask wasthen removed from the oil bath and the reaction mixture cooled toambient temperature. The stir bar was removed and the solvent wasevaporated in vacuo in a water bath at 45° C. The residue was thentreated with 50 ml of Milli-Q water and stirred for 10 minutes to give asuspension. The precipitate was collected by filtering, washed with 100ml of 2N NaHCO₃, then washed with 60 ml Milli-Q water three times, anddried over a medium frit sintered glass funnel in vacuo to give 7.15 gof white crystals (Syn. ID, WH-8-004). Yield, 95%, confirmed by ESI-MS.ESI-MS: m/z 189.2 (M+H+).

4-methyl-6-phenylpyridazin-3(2H)-one (MW01-8-008WH)

7.0 g (35 mmole) of MW01-8-004WH was placed in a 100 ml single-neckedround bottom flask followed by 9.4 g (70 mmole) of anhydrous copper (II)chloride and then 30 ml of acetonitrile to give a brown yellowsuspension. A reflux condenser was connected to the flask and a dry tubefilled with CaCl₂ was fitted to the top of the condenser. The reactionmixture was heated to reflux in an oil bath (110° C.) for 3 h. The colorof the reaction suspension changed to dark yellow once the refluxstarted. After the completion of the reaction (monitored by HPLC), theflask was removed from the oil bath and cooled to ambient temperature.The mixture was poured on to 300 g of crushed ice and stirred vigorouslyfor 10 minutes to give a gray precipitate and blue liquid. Theprecipitate was then collected by filtering (pH of the filtrate was1.5-2.0), and washed with 100 ml of a 1N HCl solution to rid the solidof any remaining copper byproducts. This is followed by washing with 100ml of Milli-Q water to get rid of the acid in the solid, and ismonitored by checking the pH value of the filtrate. The solid was washeduntil the filtrate shows a pH of 7, after approximately 5 washes. Thesolid was dried over a medium frit sintered glass funnel in vacuo togive 6.3 g of a blue gray solid. Yield was 96.7% and confirmed byESI-MS. ESI-MS: m/z 187.3 (M+H+).

3-chloro-4-methyl-6-phenylpyridazine(MW01-8-012WH)

6.0 g (32 mmole) of MW01-8-008WH and 30 ml (320 mmole) of phosphorusoxychloride were placed in a 100 ml single-necked round bottom flask.The flask was connected with a reflux condenser and a dry tube filledwith anhydrous CaCl₂ was fitted to the top of the condenser. (HCl gas isformed in the reaction so a basic solution such as NaOH may be needed toabsorb HCl in a large-scale synthesis). The reaction mixture was stirredin an oil bath (90° C.) for 2 h, then cooled to ambient temperature andpoured onto crushed ice (phosphorus oxychloride can be decomposed bywater to give HCl and H₃PO₄). The mixture was then stirred vigorouslyfor 10 minutes to give a white suspension. The suspension wasneutralized with a 2N NaOH solution until the pH of the suspension waspH=7. The precipitate was filtered, washed three times with 100 ml ofMilli-Q water and dried over a medium frit sintered glass funnel invacuo to provide 5.9 g of a light pink powder (Syn. ID, WH-8-012). Yieldwas 89.4% and confirmed by ESI-MS. ESI-MS: m/z 205.4 (M+H+).

2-(4-(4-methyl-6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine(MW01-2-151SRM)

0.82 g (4.0 mmole) of WH-8-012 was placed in a 30 ml pressure vesselfollowed by addition of 2.6 g (16.0 mmole) of 1-(2-pyrimidyl)piperazineand then 15 ml of 1-BuOH. The vessel was sealed tightly and placed intoan oil bath and stirred at 130° C. (temperature of oil bath) for 2.5days. The reaction mixture was then cooled to ambient temperature andtransferred to a single-necked flask for evaporation under reducedpressure. Removal of solvent gave rise to a brown-red residue that wastreated with 30 ml of water to give a brown sticky oil. The mixture waskept at ambient temperature overnight while the oil solidifiedgradually. The formed solid was then broken into small pieces with asteel spatula. The solid was collected by filtering and washed with 50ml of Milli-Q water three times and dried over a filter funnel in vacuoto provide 1.25 g of light yellow solid (Syn. ID, WH-8-020). Yield was94%. (Alternative separation is to use a precipitation procedure insteadof a solidification process. Solidification is a simple and cheapoperation, yet time-consuming. Precipitation is time efficient, yet morecostly than the former one. So it is up to the process chemist to decidewhich procedure to pick for the manufacture. The precipitation processis below: The oil product was dissolved completely in 10 ml of reagentgrade ethanol or acetone to form a solution. The solution was then addeddropwise to 150 ml of ice water under vigorous stirring. Light yellowsuspension was then formed gradually. The solid was collected byfiltering, washed with Milli-Q water, dried over filter funnel in vacuoto give the desired product.) The final compound was confirmed by ESI-MSand NMR. ESI-MS: m/z 333.8 (M+H+). 1H NMR (CDCl₃): d 8.380 (d, J=5.0,2H), 7.065 (d, J=7.0, 2H), 7.626 (s, 1H), 7.473 (m, 3H), 6.567 (t,J=4.5, J=5.0, 1H), 4.056 (t, J=5.0, 4H), 3.475 (t, J=5.0, 4H), 2.456 (s,3H).

C. Preparation of4,6-diphenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine (MW01-5-188WH)

4,6-diphenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine (MW01-5-188WH)was prepared by several synthetic schemes as depicted in FIG. 7 (Scheme1), FIG. 8 (Scheme 2), and FIG. 9 (Scheme 3), which were carried out asdescribed in detail herein. The various reaction schemes (Schemes 1, 2,and 3) are generally applicable to the compounds of the presentinvention and are not restricted in utility only to the preparation ofMW01-2-188WH.

Scheme 1 (FIG. 7)

3-chloro-6-phenylpyridazin-4-ol was synthesized according to theprocedure described by Coudert, P., et al. supra.

6-phenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazin-4-ol(MW01-7-121WH)

The compound was prepared from 3-chloro-4-hydroxy-6-phenylpyridazine (14g, 68 mmol). A mixture of 3-chloro-4,6-diphenylpyridazine (267 mg, 1.0mmol), 1-(2-pyrimidyl)piperazine (656 mg, 4.0 mmol) in 3 ml of 1-BuOHwas heated with stirring at 130° C. for 3 days. The solvent was removedby evaporation in vacuo the residue was treated with water to give asuspension. The solid was then filtered off, washed with water, driedover filter funnel in vacuo to give light pink solid yielding whitesolid (22.1 g, 66 mmol, 97.3%). ESI-MS: m/z 335.2 (M+H+). 1H NMR (DMSO):1H NMR (DMSO): d 8.406 (d, J=6.5, 2H), 7.740 (d, J=4.0, 2H), 7.558 (s,3H), 6.686 (t, J=4.8, J=4.4, 1H), 6.841 (s, 1H), 3.881 (s, 4H), 3.620(s, 4H), 3.776 (s, 4H).

4-chloro-6-phenyl-3-(4-pyrimidin-2-yl)piperazin-1-yl)pyridazine(MW01-6-127WH)

6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazin-4-ol (22.0 g, 66mmol) was suspended in 75 ml phosphorus oxychloride and heated withstirring at 100° C. for 3 h. After cooling to room temperature themixture was poured onto crushed ice. The mixture was then neutralizedwith NaOH solution to give white suspension. The precipitation wasfiltered off, washed with water, dried over filter funnel to providewhite solid (21.3 g, 60.3 mmol, 91.4%). ESI-MS: m/z 353.4 (M+H+). 1H NMR(CDCl₃): d 8.377 (d, J=4.5, 2H), 8.036 (d, J=7.5, 2H), 7.833 (s, 1H),7.508 (m, 3H), 6.564 (t, J=4.5, 1H), 4.073 (t, J=4.0, J=4.5, 4H), 3.672(t, J=4.0, J=4.5, 4H).

4,6-diphenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine (MW01-5-188WH)

A mixture of 3-chloro-4,6-diphenylpyridazine (267 mg, 1.0 mmol),1-(2-pyrimidyl)piperazine (656 mg, 4.0 mmol) in 3 ml of 1-BuOH washeated with stirring at 130° C. for 3 days. The solvent was removed byevaporation in vacuo the residue was treated with water to give asuspension. The solid was then filtered off, washed with water, driedover filter funnel in vacuo to give light pink solid. (320 mg, 0.81mmol, yield 81.1%). ESI-MS: m/z 395.5 (M+H+). HRMS calcd 395.1979, found395.1973; 1H NMR (CDCl₃): d 8.329 (d, J=5.0, 2H), 8.101 (d, J=7.5, 2H),7.734 (d, J=7.5, 2H), 7.655 (s, 1H), 7.509 (m, 6H), 6.530 (t, J=4.5,1H), 3.836 (t, J=4.5, J=5.0, 4H), 3.394 (t, J=5.0, J=4.5, 4H).

Scheme 2 (FIG. 8) 4,5-dihydro-6-phenyl-4-phenylpyridazin-3(2H)-one

135 ml (135 mmole) of a solution of phenylmagnesium bromide (1M) in THFwas added to a hot suspension of 6-phenylpyridazinone compound 7.8 g (45mmole) in dry toluene (50 ml). The mixture was refluxed for 8 h, leftovernight at ambient temperature, then decomposed with a saturatedsolution of ammonium chloride. The organic layer was separated, and theaqueous layer was extracted with 100 ml of ethyl acetate. The solventwas removed and the residue was crystallized from ethanol. The crystalswere collected by filtering and dried over a medium frit sintered glassfunnel in vacuo to give 5.6 g of white crystals. Yield was 50%,confirmed by ESI-MS. ESI-MS: m/z 250.1 (M+H+).

6-phenyl-4-phenylpyridazin-3(2H)-one

4.4 g (17.5 mmole) of 6-pyridazinone obtained above was placed in a 50ml single-necked round bottom flask followed by 4.7 g (35 mmole) ofanhydrous copper (II) chloride and then 20 ml of acetonitrile to give abrown yellow suspension. A reflux condenser was connected to the flaskand a dry tube filled with CaCl₂ was fitted to the top of the condenser.The reaction mixture was heated to reflux in an oil bath (110° C.) for 3h. The color of the reaction suspension changed to dark yellow once thereflux started. After the completion of the reaction (monitored byHPLC), the flask was removed from the oil bath and cooled to ambienttemperature. The mixture was poured on to 200 g of crushed ice andstirred vigorously for 10 minutes to give a gray precipitate and blueliquid. The precipitate was then collected by filtering (pH of thefiltrate was 1.5-2.0), and washed with 50 ml of a 1N HCl solution to ridthe solid of any remaining copper byproducts. This is followed bywashing with 100 ml of Milli-Q water to get rid of the acid in thesolid, and is monitored by checking the pH value of the filtrate. Thesolid was washed until the filtrate shows a pH of 7, after approximately5 washes. The solid was dried over a medium frit sintered glass funnelin vacuo to give 3.9 g of a blue gray solid. Yield was 90%, confirmed byESI-MS. ESI-MS: m/z 248.1 (M+H+).

3-chloro-6-phenyl-4-phenylpyridazine

2.0 g (8 mmole) of 6-phenylpyridazinone obtained above and 10 ml (54mmole) of phosphorus oxychloride (reagent grade, Aldrich) were placed ina 50 ml single-necked round bottom flask. The flask was connected with areflux condenser and a dry tube filled with CaCl₂ was fitted to the topof the condenser. (HCl gas is formed in the reaction so a basic solutionsuch as NaOH may be needed to absorb HCl in a large-scale synthesis).The reaction mixture was stirred in an oil bath (90° C.) for 2 h, thencooled to ambient temperature and poured onto crushed ice. (Phosphorusoxychloride can be decomposed by water to give HCl and H₃PO₄). Themixture was then stirred vigorously for 10 minutes to give a whitesuspension. The suspension was neutralized with a 2N NaOH solution untilthe pH of the suspension was pH=7. The precipitate was filtered, washedthree times with 100 ml of water and dried over a medium frit sinteredglass funnel in vacuo to provide 1.8 g of a light pink powder. Yield was85%, confirmed by ESI-MS. ESI-MS: m/z 266.4 (M+H+).

2-(4-(6-phenyl-4-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine

1.1 g (4.0 mmole) of 3-chloropyridazine obtained above was placed in a30 ml pressure vessel followed by addition of 2.6 g (16.0 mmole) of1-(2-pyrimidyl)piperazine and then 15 ml of 1-BuOH (reagent grade). Thevessel was sealed tightly and placed into an oil bath and stirred at130° C. (temperature of oil bath) for 3 days. The reaction mixture wasthen cooled to ambient temperature and transferred to a single-neckedflask for evaporation under reduced pressure. Removal of solvent gaverise to a brown-red residue that was treated with 30 ml of water to givea brown suspension. The solid was collected by filtering and washed with50 mL of water three times and dried over a filter funnel in vacuo toprovide 0.96 g of light yellow solid. Yield was 90%, ESI-MS: m/z 395.5(M+H+). HRMS calcd 395.1979, found 395.1973; 1H NMR (CDCl₃): d 8.329 (d,J=5.0, 2H), 8.101 (d, J=7.5, 2H), 7.734 (d, J=7.5, 2H), 7.655 (s, 1H),7.509 (m, 6H), 6.530 (t, J=4.5, 1H), 3.836 (t, J=4.5, J=5.0, 4H), 3.394(t, J=5.0, J=4.5, 4H).

Scheme 3 (FIG. 9)

3-chloro-6-phenylpyridazin-4-ol was synthesized according to theprocedure described by Coudert, P., et al., supra.

4,6-diphenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine (MW01-5-188WH)

A mixture of 3-chloro-4,6-diphenylpyridazine (267 mg, 1.0 mmol),1-(2-pyrimidyl)piperazine (656 mg, 4.0 mmol) in 3 ml of 1-BuOH washeated with stirring at 130° C. for 3 days. The solvent was removed byevaporation in vacuo the residue was treated with water to give asuspension. The solid was then filtered off, washed with water, driedover filter funnel in vacuo to give light pink solid. (320 mg, 0.81mmol, yield 81.1%). ESI-MS: m/z 395.5 (M+H+). HRMS calcd 395.1979, found395.1973; 1H NMR (CDCl₃): d 8.329 (d, J=5.0, 2H), 8.101 (d, J=7.5, 2H),7.734 (d, J=7.5, 2H), 7.655 (s, 1H), 7.509 (m, 6H), 6.530 (t, J=4.5,1H), 3.836 (t, J=4.5, J=5.0, 4H), 3.394 (t, J=5.0, J=4.5, 4H).

D. Preparation of4-pyridyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-6-189WH)

4-pyridyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-6-189WH) was prepared by two synthetic schemes as depicted inFIGS. 10A and 10B, which were carried out as described in detail herein.The various reaction schemes (Schemes 1 and 2) are generally applicableto the compounds of the present invention and are not restricted inutility only to the preparation of MW01-2-189WH.

Scheme 1

3-chloro-6-phenylpyridazin-4-ol was synthesized according to theprocedure described by Coudert, P., et al., supra.

6-phenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazin-4-ol(MW01-7-121WH)

This compound was prepared from 3-chloro-4-hydroxy-6-phenylpyridazine(14 g, 68 mmol). A mixture of 3-chloro-4,6-diphenylpyridazine (267 mg,1.0 mmol), 1-(2-pyrimidyl)piperazine (656 mg, 4.0 mmol) in 3 ml of1-BuOH was heated with stirring at 130° C. for 3 days. The solvent wasremoved by evaporation in vacuo the residue was treated with water togive a suspension. The solid was then filtered off, washed with water,dried over filter funnel in vacuo to give light pink solid yieldingwhite solid (22.1 g, 66 mmol, 97.3%). ESI-MS: m/z 335.2 (M+H+). 1H NMR(DMSO): 1H NMR (DMSO): d 8.406 (d, J=6.5, 2H), 7.740 (d, J=4.0, 2H),7.558 (s, 3H), 6.686 (t, J=4.8, J=4.4, 1H), 6.841 (s, 1H), 3.881 (s,4H), 3.620 (s, 4H), 3.776 (s, 4H).

4-chloro-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-6-127WH)

6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazin-4-ol 1 h (22.0 g,66 mmol) was suspended in 75 ml phosphorus oxychloride and heated withstirring at 100° C. for 3 h. After cooling to room temperature themixture was poured onto crushed ice. The mixture was then neutralizedwith NaOH solution to give white suspension. The precipitation wasfiltered off, washed with water, dried over filter funnel to providewhite solid (21.3 g, 60.3 mmol, 91.4%). ESI-MS: m/z 353.4 (M+H+). 1H NMR(CDCl₃): d 8.377 (d, J=4.5, 2H), 8.036 (d, J=7.5, 2H), 7.833 (s, 1H),7.508 (m, 3H), 6.564 (t, J=4.5, 1H), 4.073 (t, J=4.0, J=4.5, 4H), 3.672(t, J=4.0, J=4.5, 4H).

4-pyridyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-6-189WH)

Into a reaction tube were added WH-6-127 (1.4 g, 4.0 mmol), K₂CO₃ powder(1.7 g, 12.4 mmol), Pd(PPh₃)₄ (240 mg, 0.2 mmol), 4-pyridineboronic acid(664 mg, 5.4 mmol) and 20 ml of DME. Argon was then flushed through thetube for 3 min. The tube was then sealed tightly and heated withstirring at 120 degree for 24 h. After cooled down, the mixture wasfilter through a celite earth, the filtrate was then concentrated andthe residue was purified by column chromatography eluting with 1:4,Ethyl Acetate:Petroleum ether. Light yellow needle crystals wereobtained (0.65 g, 1.65 mmol, yield 41.2%). Confirmed by ESI-MS and NMR.ESI-MS: m/z 396.2 (M+H+). 1H NMR (CDCl₃): d 8.809 (d, J=6.0, 2H), 8.335(d, J=5.0, 2H), 8.090 (d, J=7.5, 2H), 7.750 (m, 6H), 6.543 (t, J=4.5,1H), 3.868 (t, J=5.0, 4H), 3.404 (t, J=5.0, 4H).

Scheme 2 4,5-dihydro-6-phenyl-4-(pyridin-4-yl)pyridazin-3(2H)-one

To a 200 ml, three-necked, round-bottomed flask equipped with a magneticstir bar, 150 ml pressure-equalizing addition funnel, reflux condenserand a glass stopper, was added 21 g (135 mmole) of 4-bromopyridine and70 of anhydrous THF. The system was oven-dried and flushed with argonbefore use. 135 ml (135 mmole) of THF solution of phenylmagnesiumbromide (1M) was placed in the pressure-equalizing addition funnel.Then, the Grignard solution was added dropwise over a period of 10minutes. After the addition, the reaction was stirred for 15 minutes forcompletion. The solution of Grignard reagent was then obtained. Asolution of 4-pyridylmagnesium bromide obtained above was added to a hotsuspension of 6-phenylpyridazinone compound 7.8 g (45 mmole) in drytoluene (50 ml). The mixture was refluxed for 8 h, left overnight atambient temperature, then decomposed with a saturated solution ofammonium chloride. The organic layer was separated, and the aqueouslayer was extracted with 100 ml of ethyl acetate. The solvent wasremoved and the residue was crystallized from ethanol. The crystals werecollected by filtering and dried over a medium frit sintered glassfunnel in vacuo to give 5.6 g of white crystals. Yield was 50%,confirmed by ESI-MS. ESI-MS: m/z 252.1 (M+H+).

6-phenyl-4-(pyridin-4-yl)pyridazin-3(2H)-one

4.4 g (17.5 mmole) of 6-pyridazinone obtained above was placed in a 50ml single-necked round bottom flask followed by 4.7 g (35 mmole) ofanhydrous copper (II) chloride and then 20 ml of acetonitrile to give abrown yellow suspension. A reflux condenser was connected to the flaskand a dry tube filled with CaCl₂ was fitted to the top of the condenser.The reaction mixture was heated to reflux in an oil bath (110° C.) for 3h. The color of the reaction suspension changed to dark yellow once thereflux started. After the completion of the reaction (monitored byHPLC), the flask was removed from the oil bath and cooled to ambienttemperature. The mixture was poured on to 200 g of crushed ice andstirred vigorously for 10 minutes to give a gray precipitate and blueliquid. The precipitate was then collected by filtering (pH of thefiltrate was 1.5-2.0), and washed with 50 ml of a 1N HCl solution to ridthe solid of any remaining copper byproducts. This is followed bywashing with 100 ml of Milli-Q water to get rid of the acid in thesolid, and is monitored by checking the pH value of the filtrate. Thesolid was washed until the filtrate shows a pH of 7, after approximately5 washes. The solid was dried over a medium frit sintered glass funnelin vacuo to give 3.9 g of a blue gray solid. Yield was 90%, confirmed byESI-MS. ESI-MS: m/z 250.1 (M+H+).

3-chloro-6-phenyl-4-(pyridin-4-yl)pyridazine

2.0 g (8 mmole) of 6-phenylpyridazinone obtained above and 10 ml (54mmole) of phosphorus oxychloride (reagent grade, Aldrich) were placed ina 50 ml single-necked round bottom flask. The flask was connected with areflux condenser and a dry tube filled with CaCl₂ was fitted to the topof the condenser. (HCl gas is formed in the reaction so a basic solutionsuch as NaOH may be needed to absorb HCl in a large-scale synthesis).The reaction mixture was stirred in an oil bath (90° C.) for 2 h, thencooled to ambient temperature and poured onto crushed ice. (phosphorusoxychloride can be decomposed by water to give HCl and H₃PO₄). Themixture was then stirred vigorously for 10 minutes to give a whitesuspension. The suspension was neutralized with a 2N NaOH solution untilthe pH of the suspension was pH=7. The precipitate was filtered, washedthree times with 100 ml of water and dried over a medium frit sinteredglass funnel in vacuo to provide 1.8 g of a light pink powder. Yield was85%, confirmed by ESI-MS. ESI-MS: m/z 268.4 (M+H+).

4-pyridyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-6-189WH)

1.1 g (4.0 mmole) of 3-chloropyridazine obtained above was placed in a30 ml pressure vessel followed by addition of 2.6 g (16.0 mmole) of1-(2-pyrimidyl)piperazine and then 15 ml of 1-BuOH (reagent grade). Thevessel was sealed tightly and placed into an oil bath and stirred at130° C. (temperature of oil bath) for 3 days. The reaction mixture wasthen cooled to ambient temperature and transferred to a single-neckedflask for evaporation under reduced pressure. Removal of solvent gaverise to a brown-red residue that was treated with 30 ml of water to givea brown suspension. The solid was collected by filtering and washed with50 mL of water three times and dried over a filter funnel in vacuo toprovide 0.96 g of light yellow solid. Yield was 90%, confirmed by ESI-MSand NMR. ESI-MS: m/z 396.2 (M+H+). 1H NMR (CDCl₃): d 8.809 (d, J=6.0,2H), 8.335 (d, J=5.0, 2H), 8.090 (d, J=7.5, 2H), 7.750 (m, 6H), 6.543(t, J=4.5, 1H), 3.868 (t, J=5.0, 4H), 3.404 (t, J=5.0, 4H).

E. Preparation ofN-(cyclopropylmethyl)-6-phenyl-4-(Pyridin-4-yl)pyridazin-3-amine(MW01-7-084WH)

A synthetic scheme for the preparation ofN-(cyclopropylmethyl)-6-phenyl-4-(pyridin-4-yl)pyridazin-3-amine(MW01-7-084WH) is depicted in FIG. 11, and synthesis was carried out asdescribed herein.

4-chloro-6-phenylpyridazin-3(2H)-one (MW01-6-093WH)

4-chloro-6-phenylpyridazin-3(2H)-one was synthesized according to theprocedure described by Coudert, P. [18].

4-chloro-2-(methoxymethyl)-6-phenylpyridazin-3(2H)-one (MW01-7-053WH)

A mixture of chloropyridazinone 1 (25.5 g, 0.12 mol),4-N,N-dimethylaminopyridine (0.20 g) and i-Pr₂NEt (26.7 g, 0.21 mol) inanhydrous CH₂Cl₂ (300 mL) was stirred at 0° C. (ice bath) for 30 min.Methoxymethyl chloride (25 g, 0.31 mol) was added and the mixture wasstirred at 0° C. for 1 h and then allowed to warm to room temperature.The reaction was stirred at room temperature till complete. The solventwas then removed in vacuo, the residue was treated with water, washedwith dilute Na₂CO₃ solution and extracted with EtOAc. The organic layerwas dried over anhydrous Na₂SO₄, filtered and evaporated. The residuewas then purified by recrystallization from 95% ethanol to give 20.1light yellow solid. Yield 66.9%.

6-phenyl-4-(pyridin-4-yl)pyridazin-3(2H)-one (MW01-7-069WH)

The protected pyridazinone MW01-7-053WH (1.0 equiv.) was mixed witharylboronic acid (1.37 equiv.), Pd(PPh₃)₄ (0.05 equiv.) and K₂CO₃ (3.1equiv) and 200 mL of DME in a 350 ml of pressure vessel, flushed withargon for 3 min, and the mixture was then stirred and refluxed (oilbath, 120° C.) until the starting material had disappeared. Aftercooling, the solution was concentrated to dryness under reducedpressure, the residue was treated with water and filtered off. Thefilter cake was washed with water over filter funnel and then used fornext step directly. The residue obtained above was dissolved in 200 mlof EtOH, 6 N HCl (200 mL) was added and the reaction mixture wasrefluxed (oil bath, 120° C.) for 6 h, then it was allowed to cool toroom temperature, and concentrated to dryness under reduced pressure.The residue was neutralized with dilute NaOH solution. The suspensionwas then filtered off, washed with water and dried over a filter funnel.Recrystallization from 90% ethanol provided brown yellow solid. Yield80.4%. ESI-MS: m/z 294.3 (M+H+)

3-chloro-6-phenyl-4-(pyridin-4-yl)pyridazine (MW0′-7-076WH)

3-chloro-6-phenyl-4-(pyridin-4-yl)pyridazine (MW01-7-076WH) (66 mmol)was suspended in 75 ml phosphorus oxychloride and heated with stirringat 100° C. for 3 h. After cooling to room temperature the mixture waspoured onto crushed ice. The mixture was then neutralized with NaOHsolution to give white suspension. The precipitation was filtered off,washed with water, dried over filter funnel to yielding a light yellowsolid. ESI-MS: m/z 268.4 (M+H+).

N-(cyclopropylmethyl)-6-phenyl-4-(pyridin-4-yl)pyridazin-3-amine(MW01-7-084WH)

A mixture ofN-(cyclopropylmethyl)-6-phenyl-4-(pyridin-4-yl)pyridazin-3-amine(MW01-7-084WH) (0.5 mmol), C-Cyclopropyl-methylamine (2.0 mmol) in 3 mlof 1-BuOH was heated with stirring at 130° C. for 7 days. The solventwas removed by evaporation in vacuo, the residue was treated with waterto give a suspension. The solid was then filtered off, washed withwater, then 1:3, Ethyl Acetate:Petroleum ether, dried over filter funnelin vacuo yielding gray solid. ESI-MS: f/z 330.4 (M+H+).

F. Preparation of3-(4-methylpiperazin-1-yl)-6-phenyl-4-(pyridin-4-yl)pyridazine(MW01-7-085WH)

A mixture of 3-chloro-6-phenyl-4-(pyridin-4-yl)pyridazine (MW01-7-076WH)(0.5 mmol), 1-methyl-piperazine (2.0 mmol) in 3 ml of 1-BuOH was heatedwith stirring at 130° C. for about 7 days. The solvent was removed byevaporation in vacuo the residue was treated with water to give asuspension. The solid was then filtered off, washed with water, then1:3, Ethyl Acetate:Petroleum ether, dried over filter funnel in vacuo toyield a brown solid. ESI-MS: m/z 332.2 (M+H+). A synthetic reactionscheme for the preparation of3-(4-methylpiperazin-1-yl)-6-phenyl-4-(pyridin-4-yl)pyridazine(MW01-7-085WH) is depicted in FIG. 12.

G. Preparation of 4,6-diphenyl-3-piperazinylpyridazine (MW01-7-133WH)

A synthetic reaction scheme for the preparation of4,6-diphenyl-3-piperazinylpyridazine (MW01-7-133WH) is depicted in FIG.13, and synthesis was carried out as described herein. The compound wasprepared from 3-chloro-4,6-diphenylpyridazine (533 mg, 20 mmole) in thesame manner as described for MW01-7-057WH, yielding light yellow solid(550 mg, 17.4 mmole, yield 86.9%). ESI-MS: m/z 317.3 (M+H+). 1H NMR(CDCl₃): d 8.086 (d, J=7.5, 2H), 7.705 (d, J=7.5, 2H), 7.619 (s, 1H),7.498 (m, 6H), 3.318 (d, J=4.0, 4H), 2.932 (d, J=4.0, 4H) 1.896 (s, 1H).

H. Preparation of2-(4-(6-phenyl-4-(Piperidin-1-yl)pyridazin-3-yl)piperazin-1-yl)pyrimidine(MW01-7-107WH)

A synthetic reaction scheme for the preparation of2-(4-(6-phenyl-4-(piperidin-1-yl)pyridazin-3-yl)piperazin-1-yl)pyrimidine(MW01-7-107WH) is depicted in FIG. 14, and synthesis was carried out asdescribed herein. The compound was prepared from MW01-6-127WH (200 mg,0.57 mmole) in the same manner as described for MW01-7-057WH, yieldinglight yellow solid (220 mg, 0.55 mmole, yield 96.3%). ESI-MS: m/z 402.5(M+H+).

I. Preparation of6-methyl-4-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-7-057)

A synthetic reaction scheme for the preparation of6-methyl-4-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine(MW01-7-057) is depicted in FIG. 15, and synthesis was carried out asdescribed herein. A mixture of 3-chloro-6-methyl-4-phenylpyridazine (100mg, 0.5 mmol), 1-(2-pyrimidyl)piperazine (400 mg, 2.0 mmol) in 3 ml of1-BuOH was heated with stirring at 130° C. for 7 days. The solvent wasremoved by evaporation in vacuo the residue was treated with water togive a suspension. The solid was then filtered off, washed with water,then 1:3, Ethyl Acetate:Petroleum ether, dried over filter funnel invacuo to give light yellow solid (68 mg, 0.20 mmol, yield 41.7%).Purity >95%; ESI-MS: m/z 333.1 (M+H+). 1H NMR (CDCl₃): d 8.310 (d,J=5.0, 2H), 7.678 (d, J=7.5, 2H), 7.476 (m, 3H), 7.119 (s, H), 6.509 (t,J=4.5, 1H), 3.785 (t, J=4.5, J=5.0, 4H), 3.277 (t, J=4.5, J=5.0, 4H),2.669 (s, 3H).

Example 2 Assays for Confirming Activity of Pyridazine Compounds

The following assays can be used to confirm the activity of thepyridazine compounds. Cell culture assays. Cell-based assays of theconcentration-dependent activity of a compound of the invention will beconducted using methods previously described (Mirzoeva et al., J MedChem 45:563-566, 2002). BV-2 mouse microglial cells (1.25×10⁴ cells/wellin a 48-well plate) will be cultured for one day in αMEM mediacontaining 10% fetal bovine serum (FBS), and then treated in serum-freemedia for 16 hrs with either control buffer or the standard glialactivating stimulus lipopolysaccharide (LPS, from Salmonellatyphimurium; 100 ng/ml final concentration) in the presence of diluentor compound. Stock solutions (20 mM) of compounds will be prepared indimethylsulfoxide (DMSO). Solutions for cell treatments will be preparedby dilution of stock solutions into serum-free media immediately beforeadding to the cells. Control wells will contain the same finalconcentration of DMSO as the compound-containing wells. It has beenpreviously determined that this concentration of DMSO is not toxic tothe cells (Mirzoeva et al., Brain Res. 844:126-134, 1999). Theaccumulation of nitrite, the stable metabolite of nitric oxide (NO),will be measured in BV-2 conditioned media by the Griess assay aspreviously described (Mirzoeva et al., Brain Res. 844:126-134, 999;Mirzoeva et al., J Med Chem 45:563-566, 2002). Levels of IL-1β in celllysates and TNFα in conditioned media will be measured by ELISA(Biosource International) as per the manufacturer's instructions. Celllysates will be analyzed by Western blots as described (Mirzoeva et al.,J Med Chem, 2002) to determine the levels of inducible nitric oxidesynthase (iNOS), cyclooxygenase-2 (COX-2) and apolipoprotein E (apoE).For apoE measurements, rat primary mixed glia will be prepared andstimulated with human oligomeric Aβ₁₋₄₂ (10 μM) as previously described(Mirzoeva et al., 2002, supra). Antibodies and dilutions used forWestern blots will be as follows: anti-COX-2 (1:1000, Santa Cruz),anti-iNOS (1:1000, Transduction Laboratories), anti-apoE (1:1000).Antibody against M-actin (1:500,000 dilution, Sigma) will be used toconfirm equal protein loading among the samples. In vivo efficacystudies in mice. The study design and treatment paradigm forintracerebroventricular (ICV) infusion of human oligomeric Aβ₁₋₄₂ intothe mouse will be as described previously (Craft et al., Neurobiol Aging25:1283-1292, 2004b), except that compound administration will be bymouth. Female C57Bl/6 mice (Harlan) weighing 20-25 g (3-4 months old)will be housed in a pathogen free facility under an approximate 12 h/12h dark and light cycle and they will have access ad libitum to food andwater.

Mice will be administered by oral gavage either test compound (2.5mg/kg/day) or solvent control (10% DMSO) in a 0.5% (w/v)carboxymethylcellulose suspension. Once per day treatment will begin atday 21 after start of Aβ ICV infusion and continue for 14 days.Beginning at day 50 after start of Aβ ICV infusion, the Y maze test ofspontaneous alternation will be used to evaluate hippocampus-dependentspatial learning as described previously (Craft et al., J Mol Neurosci24:115-122, 2004a). Briefly, each mouse will be placed in the “start”arm and then released to choose one of the two other arms. The mousewill be blocked from exiting the chosen arm for 30s then they will beplaced back in the start arm and released again to choose one of the twoother arms. If the second choice is different from the first one, themouse will be scored as alternating. Mice will be tested for 10 dayswith one trial per day, and a mean percent alternation will becalculated for each mouse. At day 60 after start of Aβ ICV infusion,mice will be anesthetized with pentobarbital (50 mg/kg) and perfusedwith a HEPES buffer (10 mM, pH 7.2) containing a protease inhibitorcocktail (1 μg/ml leupeptin, 1 μM dithithreitol, 2 mM sodium vanadate, 1μM phenylmethylsulphonylfluoride). The brain will be removed andlongitudinally bisected as described previously (Craft et al.,Neurobiolo Aging 25:1283-1292, 2004b). The right half of the brain willbe fixed in 4% (v/v) paraformaldehyde and paraffin-embedded forhistology. The hippocampus will be dissected from the left half of thebrain and snap-frozen for subsequent biochemical evaluation. Hippocampalextract supernatants will be prepared by dounce and sonication in theHEPES buffer containing a protease inhibitor cocktail, followed bycentrifugation as described (Craft et al., 2004b, supra).

Levels of IL-1β and TNFα in hippocampal supernatants will be measured byELISA (Biosource International) per the manufacturer's instructions.S100B levels in hippocampal supernatants will be measured by aeuropium-based ELISA essentially as previously described (Van Eldik andGriffin, Biochem Biophys Acta 1223:398-403, 1994). Synaptophysin levelsin hippocampal supernatants will be quantified by ELISA following theprocedure described previously (Craft et al, 2004b, supra). PSD-95levels will be determined by Western blots using anti-PSD-95 antibodies(1:100,000 dilution; Upstate Biotechnology) as described (Craft et al.,2004b).

Immunohistochemical detection of activated astrocytes and microglia willbe performed on 10 μm sections as described previously (Craft et al,2004b, supra), with anti-GFAP (1:1500; Sigma) and anti-F4/80 (1:100;Serotek) antibodies, respectively, using the mouse on mouse orVectastain Universal Elite ABC immunodetection kits (Vector/Novocastra)and development with diaminobenzidine (DAB) substrate. Cell bodies willbe manually counted in the hippocampus of three GFAP and F4/80 labeledsections positioned at −1.8, −2.1, and −2.3 mm from bregma. Aβimmunohistochemistry will be done with a rabbit anti-human Aβ antibodyas previously described (Craft et al., 2004b, supra). Cell counts andamyloid plaque counts will be determined by two blinded observers andamyloid plaque area will be determined as previously described (Craft etal., 2004b, supra). Peroxynitrite-mediated neuronal damage will bemeasured with an anti-nitrotyrosine antibody (1:125; Chemicon), usingthe Vectastain Rabbit Elite ABC kit. For nitrotyrosine cell counts, allDAB-stained cell bodies in the neuronal layers of the hippocampus andsubiculum will be counted on three sections roughly adjacent to thoseused for F4/80 and GFAP analysis, as described (Craft et al., 2004b,supra).

In vitro stability, oral bioavailability and brain uptake. The stabilityof compounds (1 μM) in a standard incubation with rat liver microsomes(BD Biosciences) and an NADPH-regenerating system will be done at 37° C.for 30 and 120 min. Reactions will be stopped by acetonitrile, and thereaction mixture will be centrifuged at 16 000×g for 10 min. 10 μl ofthe supernatant will be analyzed by calibrated HPLC to quantify thepercentage of the initial amount of compound remaining after theincubation. The HPLC system (Dionex Corp., Sunnyvale, Calif.) includes aDionex P480 pump, a Phenomenex Luna C18 column (250×2.0 mm, 5 μm) with aguard column (Phenomenex, Torrance, Calif.) and a Dionex UVD340UUltraviolet (UV) detector. The mobile phase will consist of 0.1% formicacid as reagent A and 0.08% formic acid/water in 80% acetonitrile asreagent B, at a flow rate of 0.2 ml per minute. The gradient willconsist of the following linear and isocratic gradient elution changesin reagent B: isocratic at 60% from 0 to 5 min, 60% to 90% from 5 to 39min, isocratic at 90% until 44 min. Peak quantification will be donebased on absorption measured at 260 nm relative to a standard curveobtained by using serial dilutions of the compound.

To estimate oral bioavailability (concentration of compound in the bloodas a function of time after oral administration) and to gain insightinto potential brain uptake, a compound (2.5 mg/kg) will be administeredto mice by oral gavage in a 0.5% (w/v) carboxymethylcellulosesuspension. At 5, 15, 60 and 120 min after compound administration, theanimals will be anesthetized with pentobarbital (50 mg/kg). Blood willbe harvested by intracardiac puncture, collected in heparinized tubes,and plasma will be obtained by centrifugation. Mice will be perfusedwith a HEPES buffer (10 mM, pH 7.2) containing a protease inhibitorcocktail (1 μg/ml leupeptin, 1 μM dithithreitol, 2 mM sodium vanadate, 1μM phenylmethylsulphonylfluoride), and brains will be removed andweighed. Brain homogenates will be prepared by dounce and sonication inthe HEPES buffer containing a protease inhibitor cocktail. Brainhomogenates will be centrifuged at 12000×g for 10 minutes and thesupernatant acidified by diluting 1:3 with 0.1% formic acid (Fluka).Solid phase extraction followed by HPLC analysis will be used toquantify the amount of compound in brain supernatants. Briefly,cartridges (Sep-Pak® C18, Waters) will be conditioned with 1 ml ofacetonitrile (HPLC grade, EMD Biosciences) and equilibrated with 1 ml ofwater. A structural analog of the compound will be used as an internalstandard. The acidified brain supernatant will be added to the cartridgefollowed by a 1 ml wash with 30% acetonitrile. The compound will beeluted from the cartridge using 80% acetonitrile. The eluate will beevaporated to dryness, reconstituted in 0.08% formic acid/water in 80%acetonitrile and analyzed by HPLC using the following gradient inreagent B: 0% to 60% from 2 to 5 min, isocratic at 65% until 7 min, 65%to 80% from 7 to 12 min, isocratic at 80% until 15 min, 89% to 100% from15 to 18 min and isocratic at 100% until 23 min. Plasma samples will bedeproteinized in 0.1M perchloric acid and centrifuged at 12000×g for 10min. The supernatant will be neutralized with 1M NaOH then extractedwith dichloromethane, and the layers separated at 3000×g for 5 min. Theorganic phases from three successive extractions will be pooled and thenevaporated to dryness under reduced pressure. The dried residue will bereconstituted in 50 μl of reagent B, and 10 μl of the reconstitutedmaterial will be analyzed by HPLC using the gradient described above forbrain supernatants.

Suppression of CNS versus peripheral inflammation. Mice will beadministered by oral gavage of compound (2.5 mg/kg/day) or diluent (10%DMSO) in a 0.5% (w/v) carboxymethylcellulose suspension once daily fortwo weeks. After the last administration, mice will be injectedintraperitoneally (i.p) with 10 mg/kg of LPS. Control mice will beinjected with saline. Six hours after the LPS challenge, mice will beanesthetized with pentobarbital (50 mg/kg) and blood will be drawn byintracardiac puncture, allowed to clot, and centrifuged for serumpreparation. Brains will be removed and processed as described above.Levels of IL-1β and TNFα in brain supernatants and serum will bemeasured using a MSD multiplex assay per the manufacturer's instructions(Meso Scale Discovery, Gaithersburg, Md.).

Liver toxicity after chronic in vivo administration of Compound. Micewill be administered by oral gavage either test compound (2.5 mg/kg/day)or diluent (10% DMSO) in a 0.5% (w/v) carboxymethylcellulose suspensiononce daily for two weeks. Mice will be anesthetized and sacrificed asdescribed above. Livers will be removed, fixed in 4% (v/v)paraformaldehyde and paraffin-embedded for histology. To assesshistological toxicity, 4 μm liver sections will be stained withhaematoxylin and eosin. Two independent observers blinded to thetreatment groups will perform microscopic assessment of the tissue forinjury.

Morris Water Maze. This test is based on the swimming maze test forspatial memory (Morris, Learn Mot 12:239-260, 1981; J Neurosci Methods11:47-60, 1984) and takes advantage of the natural swimming ability ofrodents and the ease of manipulating cues around the maze. In this task,a mouse is placed in a pool of liquid that is made opaque by theaddition of non-toxic tempera powdered paint. The mouse then swims untilan escape platform (hidden just under the surface of the water) isfound. Finding the platform enables the mouse to escape from the waterand therefore is positively reinforced. When the platform is kept in thesame position, the animal quickly learns to use distal cues to locatethe position of the platform, even if the mouse is placed in the pool atdifferent starting positions. The experimental protocol for the Morrismaze test is as described in Ohno et al, (Eur. J. Neurosci. 2006, 23(8):2235-40; Learn Mem 2005, 12(3): 211-5). Briefly, the pool is 1.2 m indiameter and made of white metal. The water is maintained at 25±1° C.and is made opaque with nontoxic white paint to hide the square, whiteescape platform (10 cm×10 cm). During training, the platform issubmerged (1 cm) below the water surface and remains in the sameposition to avoid quandrant biases. The mice receive six trials per dayfor 4 days (3 blocks of two trials; 1 min intertrial intervals, 1-hourinterblock intervals). The mouse is placed into the water facing thewall of the pool and is allowed to search for the platform. The startingposition varies among four locations in a pseudorandom manner for eachtrial. The trial ends when an animal climbs onto the platform or when amaximum of 60 sec has elapsed. The mouse is placed on the platform for60 sec before and after each trial. At the end of the training, all miceare given a probe test with the platform removed from the pool. Thebehaviour of the mouse is recorded by a video camera and analyzedcomputationally for several parameters such as latency to finding theplatform, total distance traveled, and percent of time spent in thetarget quadrant.

At post-operative day 60 mice will be anesthetized and perfused with aHepes buffer containing a protease inhibitor cocktail. The brains arethen removed and longitudinally bisected. The right half of the brain isfixed in a paraformaldehyde/phosphate buffer solution and embedded inparaffin for histological examination, while the hippocampus is isolatedfrom the left hemisphere and snap frozen for biochemical evaluation ofendpoints.

Example 3 Efficacy in the Tg6799 5X FAD Mouse Model

MW01-2-151SRM will be tested in the Tg6799 mouse at 5, 10 and 25 mg/kg.As above, neuroinflammation and synaptic dysfunction biochemicalendpoints and Y-maze behavioral endpoint will be determined. A higherdose is proposed based on the start of administration to animals thatare already showing signs of pathology based on characterization ofstrain. More animals needed for significance are compared to theinfusion model and longer time due to required expansion of colony viabreeding.

Example 4 Selection of Lead Drug Compound

The following eight compounds were synthesized: MW01-4-179LKM;MW01-2-151SRM; MW01-7-107WH; MW01-6-189WH; MW01-7-084WH; MW01-7-085WH7)MW01-7-133WH; and MW01-7-057WH (See FIGS. 1 to 15 and Example 1).

A. The compounds were tested in glial cell-based assays forconcentration-dependent suppression of neuroinflammation endpoints(nitric oxide, IL-1β). All eight compounds inhibited LPS-induced IL-1βproduction in BV-2 microglia cells in a concentration-dependent manner.Most compounds were also selective, in that they did not inhibitproduction of nitric oxide (NO). The lack of an effect on NO productionwas further validated by showing no effect on up-regulated levels ofiNOS. No effect over the same concentration range was seen onup-regulation of COX-2. The following were selective compounds:MW01-2-151SRM; MW01-4-179LKM; MW01-6-189WH; MW01-7-084WH; MW01-7-085WH;MW01-7-133WH; and MW01-7-057WH. One compound, MW01-7-107WH, wasnon-selective in that it also inhibited production of NO, iNOS and COX-2over the same concentration ranges. (See FIGS. 16 to 23 showing theresults of the cell-based activity of MW01-2-151SRM; MW01-6-189WH;MW01-4-107WH; MW01-4-179LKM; MW01-7-084WH; MW01-7-085WH; MW01-7-133WH;and MW01-7-057WH in BV-2 microglial cells.)B. Testing of Compounds in the human Aβ infusion mouse model forsuppression of neuroinflammation and neuronal dysfunction biochemicalendpoints (IL-1β, S100B, synaptophysin). Specifically, the followingfive active compounds were tested in vivo: MW01-2-151SRM; MW01-6-189WH;MW01-7-084WH MW01-7-085WH; and MW01-7-057WH. The best compounds in vivowere MW01-2-151SRM and MW01-6-189WH. These two compounds blocked theup-regulation of IL-1β and S100B, and prevented the loss of PSD-95.MW01-2-151SRM also prevented the loss of synaptophysin. MW01-6-189WHshowed a trend toward preventing the synaptophysin loss; however,statistical significance was not reached due to limitations in samplesize. MW01-7-084WH and MW01-7-085WH blocked the upregulation of IL-1βand S100B, and prevented loss of PSD-95. They were not as effective asMW01-2-151SRM in preventing the synaptophysin loss. MW01-7-057WH blockedS100B upregulation and synaptophysin loss, but did not block IL-1βupregulation or PSD-95 loss. (See FIGS. 24 to 28 showing the results ofin vivo activity of MW01-2-151SRM; MW01-6-189WH; MW01-7-084WH;MW01-7-085WH; and MW01-7-057WH in the Aβ infusion mouse model.)C. The lead compounds were tested in the human Aβ infusion mouse modelusing the Y-maze behavioral assay at 1.25, 2.5, 5, and 10 mg/kg.Neuroinflammation biochemical endpoints (hippocampus levels of IL-1α,TNFα) are based on proposed mechanism of action, and a synapticdysfunction biochemical endpoint (hippocampus levels of synaptophysin)is used, as well as a Y-maze behavioral endpoint. MW01-2-151SRM,MW01-6-189WH, and MW01-7-057WH were significantly effective inpreventing the Y-maze behavioral deficit brought about by human Aβinfusion. MW01-7-084WH and MW01-7-085WH showed a trend toward preventingthe Y maze behavioral deficit.

Example 5 hERG Channel Inhibition Assays and Cardiac QT Interval Assays

Compounds have been screened for hERG (human ether-a-go-go) potassiumion channel binding and inhibition in order to eliminate early in theprocess any compounds with high potential to induce prolongation ofcardiac QT interval in later studies due to off-target toxicities. ThehERG channel conducts rapidly activating delayed rectifier potassiumcurrents that critically contribute to cardiac repolarization. Mutationsin the hERG channel gene and drug-induced blockade of the currents havebeen linked to delayed repolarization of action potentials resulting inprolonged QT interval (Finlayson et al., 2004; Recanatini et al., 2005;Roden, 2004). QT prolongation is considered a significant risk factoragainst cardiac safety of new drugs. Therefore, consideration of cardiacsafety early in the development process by testing for hERG channelinhibition provides an efficient and predictive means to assesspotential compound cardiac safety liabilities. In addition, the FDA(USA) is considering this as an approval criteria in the future and hasspecific recommendations. The assays done to date have been by acommercial service (MDS PharmaService).

The initial assay is a radioligand binding assay that tests the abilityof the test compound to compete with ³H-astemizole (a reference standardthat binds to hERG channels with nM affinity) for binding to recombinanthERG channels stably expressed on human HEK-293 cells. This cell linewas chosen because it is of human origin, has been fully characterizedwith regard to hERG electrophysiology and pharmacology and displays theexpected characteristics of I_(Kr) current as well as expectedpharmacological sensitivities, and is easy to maintain in culture (Zhouet al., J. Gen Physiol. 1998, 111(6): 781-94). A single concentration(10 μM) of test compound is assayed, and % inhibition of ³H-astemizolebinding is calculated. Generally, any compounds that show >50%inhibition are tested further in the hERG channel activity assay. Thisis usual for medium throughout screens but is not recommended in the FDAdocument and tends to give false positives, as evidenced by the resultsreported below.

The hERG channel activity inhibition assay provides whole cellelectrophysiological data about compound effects on the hERG K⁺ channelfunction. Whole cell patch clamp methodology is generally considered tobe the gold-standard determination of ion channel activity, rather thansimply measuring channel binding. The standard testing procedure is touse 3 to 5 concentrations of compound at log dilutions with eachconcentration tested in triplicate (three cells). This allows a balancebetween achieving a reasonably accurate IC₅₀ measurement against a broadconcentration range, and reducing cell attrition that would occur duringmore protracted experiment durations. After completion of compounddose-response procedures, a known hERG channel inhibitor, such asastemizole, is applied as a positive control.

Compounds which exhibit inhibition of hERG channel activity are verifiedas positives (the hERG channel activity assay can give false positivesand false negatives) by testing in vivo for prolongation of cardiac QTinterval. The QT interval studies are performed by evaluating compoundsfor effects on QT interval in Lead II electrocardiograms measured inanesthetized guinea pigs (Hirohashi et al., 1991, Arzneim.-Forsch./DrugRes 41:9-18), one of the species recommended in the FDA white paper.Vehicle or compound is administered orally at 15 mg/kg (dosing volume of10 ml/kg) to groups of male guinea pigs (weighing 330-350 g), with 5animals per group. This dose corresponds approximately to 20-fold thetherapeutic dose by taking into account the body surface area of theanimals. Heart rate, arterial blood pressure, and QT intervals aremeasured at baseline, and at 15, 30, 45, and 60 min after compoundadministration. Sotalol administered iv at 0.3 mg/kg serves as thepositive control compound. The QT intervals are corrected for changes inheart rate using both Bazett's and Fridericia's formulae. Any increasein QT interval values over baseline values exceeding the upper 95%confidence limit of the mean changes at the corresponding time point inthe vehicle-treated control group for two consecutive observation timesindicates significant QT interval prolongation in the individuallytreated animals. This functional testing in early discovery provides arapid and cost-effective method to better anticipate and eliminatecompounds that may have adverse QT prolongation potential in humans.

Calculations of Amount of Compound Needed:

Competition binding assay: 1-2 mgPatch clamp assay: 1-2 mgQT interval assay: 5 mg/animal/dose=25 mg per assay at 15 mg/kg dose

Because the ex vivo activity assays are subject to false positives andnegatives, it is considered better to complete studies of in vivo QTinterval assay following the guidelines of the FDA position paper.

Results: Competition Inhibition Assay:

MW01-5-188WH, MW01-2-151SRM, and MW01-6-127WH were tested at 10 μMconcentration.

MW01-5-188WH showed 91% inhibition at 10 μM. MW01-2-151SRM andMW01-6-127WH were negative, showing only 8% and 19% inhibition,respectively.

Patch Clamp Inhibition Assay:

MW01-2-151SRM and MW01-6-189WH were tested at three concentrations (0.1,1, 10 μM). These compounds showed minimal inhibition, with IC₅₀ valuesof 4.81 μM for MW01-6-189WH and 9.21 μM for MW01-2-151SRM.

Cardiac QT Interval Prolongation Assay

A summary of the results as well as the materials and methods are setout below.

Summary

A test substance (e.g., MW01-2-151SRM) was evaluated for possibleeffects on QT interval in Lead II electrocardiogram measure inanesthetized guinea pigs. The QT intervals (QTc) were corrected forchanges in heart rate using both Bazett's and Fridericia's formulae. Anyincrease in QTc values over baseline values exceeding the upper 95%confidence limit of the change at corresponding time point in thevehicle-treated control group for 2 consecutive observation timesindicates significant QTc prolongation in the individually treatedanimals. The test substance at 15 mg/kg PO did not cause any significantprolongation in QTc interval in all of the 5 treated animals during the60-minute period post-dosing (FIGS. 29 and 31). On the other hand,intravenous administration of sotalol at 0.3 mg/kg caused significantprolongation in QTc interval in all (5.5) animals (FIGS. 30 and 32). Theresults reached similar conclusion by using either Bazett's orFridericia's formula for QT correction.

MW01-5-188WH and MW01-2-151SRM were administered PO at 15 mg/kg to 5guinea pigs (330-350 g weight). QT intervals were obtained at baselineand at 15 min, 30 min, 45 min, and 60 min after compound administration.Neither compound increased cardiac QT interval above the mean+2SD ofcorresponding values in the vehicle control group. There were also nosignificant effects on mean blood pressure or heart rate after compoundadministration.

Example data for MW01-5-188WH are shown in FIG. 33. The positive controlcompound, sotalol, induces a significant increase in cardiac QTcinterval.

Materials and Methods

The test substance was dissolved in 2% Tween 80 and administered by oraladministration. The substance was treated at 15 mg/kg with a dosingvolume of 10 ml/kg with a dosing volume of 10 ml/kg. Duncan Hartleyderived guinea pigs provided by MDS Pharma Services—Taiwan Ltd wereused. Sotalol was obtained from Sigma, USA.

Groups of guinea pigs (weighing 330-350 g) with 5 animals each wereemployed. The animals were anesthetized with urethane (1500 mg/kg, IVbolus injection in a volume of 5 ml/kg) and breathed spontaneously. LeadII ECG was obtained with subdermal needle electrodes and ECG signalconditioner. Heart rate was measured with a pulse rate tachometer. Thecarotid artery was cannulated with a catheter that was connected to apressure transducer and a pressure processor for measurements ofarterial blood pressure (BP). Five parameters [HR, Q-T Interval,QTc(Bazett's), QTc(Fredericia's), BP] were recorded and displayed on aDigital Acquisition Analysis and Archive System (PO-NE-MAH, Inc. USA).QTC intervals were obtained by correction for heart rate changes usingBazett's and Fridericia's formulae. Increase in QTc interval inindividual treated guinea pigs that lies outside the upper limit of 95%confidence limits (mean±SD) of the changes for the vehicle-treatedcontrol at corresponding time points for two consecutive times isconsidered significant.

Example 6 Acute and Chronic Toxicity Assays

Liver toxicity is an especially important initial consideration fororally administered compounds, as the liver is the major site of initialdrug metabolism and is critical to overall metabolism and homeostasis ofan animal. Liver injury is also a component of idiopathic tissue injuryseen in certain chronically administered drugs. Therefore, it isimportant to do initial assessments of liver toxicity after oraladministration of compounds to mice.

Methods:

A standard approach is to test compounds in two initial in vivo toxicityassays: an acute, escalating-dose paradigm and a chronic, therapeuticdose regimen. For the escalating-dose, acute toxicity assays, mice (5per experimental group) are administered either compound or vehicle in0.5% carboxymethylcellulose (alternatively, castor oil or sesame oil canbe used) by oral gavage once daily for 3 days. Standard compound dosesare 3.1, 12.5, and 50 mg/kg; the highest dose is 20× a therapeutic dose.On the 4^(th) day, mice are sacrificed and the liver harvested and fixedfor histology. Paraffin-embedded, hematoxylin & eosin (H&E)-stainedsections of liver tissue are analyzed microscopically for injury by twoindividuals blinded to the treatment groups. A semi-quantitativehistological scoring system from 0 (best) to 9 (worst) is applied thatconsiders architecture features (normal to extensive fibrosis), cellularfeatures (normal to extensive edema and widespread necrosis), and degreeof inflammatory infiltrate (normal to extensive infiltrate). For eachacute toxicity assay, 15 mg of compound is required.

For the therapeutic dose, chronic toxicity assays, mice (5 perexperimental group) are administered either compound or vehicle in 0.5%carboxymethylcellulose by oral gavage once daily for 2 weeks at atherapeutic dose of 2.5 mg/kg/day. After two weeks of treatment, miceare sacrificed and liver toxicity analyzed as described above. For eachchronic toxicity assay, 5 mg of compound is required.

Results:

The results of the toxicity study are shown in FIG. 34.

MW01-5-188WH has been tested in the acute, escalating-dose assay and thechronic, therapeutic dose assay. There was no histological evidence oftissue toxicity at the lower doses but some vacuolisation was observedat the 50 mg/kg dose.

MW01-2-151SRM has been tested in the chronic, therapeutic dose assay.There was no histological evidence of tissue toxicity; no differenceswere seen by histology in livers from mice treated with vehicle or withcompound.

MW01-6-189WH has been tested in the chronic, therapeutic dose assay.There was no histological evidence of tissue toxicity; no differenceswere seen by histology in livers from mice treated with vehicle or withcompound.

MW01-5-188WH was tested in the chronic, therapeutic dose assay. Inparticular, mice were administered by oral gavage either MW01-5-188WH(2.5 mg/kg) or diluent (10% DMSO) in a 0.5% (w/v) carboxymethylcellulosesuspension once daily for 2 weeks. Mice were anesthetized and killed asdescribed above. Livers were removed, fixed in 4% (v/v)paraformaldehyde, and paraffin-embedded for histology. To assesshistological toxicity, 41m liver sections were stained with hematoxylinand eosin. Two independent observers blinded to the treatment groupsperformed microscopic assessment of the tissue for injury. Histologicalassessment of liver tissue showed that oral administration ofMW01-5-188WH at 2.5 mg/kg daily for 2 weeks did not induce any indicesof hepatotoxic tissue injury compared with mice treated with thediluent.

Example 7

In vitro stability, oral bioavailability, and brain uptake. Thestability of MW01-5-188WH (1 μM) in a standard incubation with rat livermicrosomes (BD Biosciences, Bedford, Mass.) and an NADPH-regeneratingsystem was done at 37° C. for 30 and 120 min. Reactions were stopped byacetonitrile, and the reaction mixture was centrifuged at 16,000 μg for10 min. Ten microliters of the supernatant were analyzed by calibratedHPLC to quantify the percentage of the initial amount of MW01-5-188WHremaining after the incubation. The HPLC system (Dionex, Sunnyvale,Calif.) includes a Dionex P680 pump, a Phenomenex (Torrance, Calif.)Luna C18 column (250×2.0 mm; 5 μm) with a guard column, and a DionexUVD340U ultraviolet detector. The mobile phase consisted of 0.1% formicacid as reagent A and 0.08% formic acid/water in 80% acetonitrile asreagent B at a flow rate of 0.2 ml per minute. The gradient consisted ofthe following linear and isocratic gradient elution changes in reagentB: isocratic at 60% from 0 to 5 min, 60-90% from 5 to 39 min, isocraticat 90% until 44 min. Peak quantification was done based on absorptionmeasured at 260 nm relative to a standard curve obtained by using serialdilutions of MW01-5-188WH. To estimate oral bioavailability(concentration of compound in the blood as a function of time after oraladministration) and to gain insight into potential brain uptake,MW01-5-188WH (2.5 mg/kg) was administered to mice by oral gavage in a0.5% (w/v) carboxymethylcellulose suspension. At 5, 15, 60, and 120 minafter compound administration, the animals were anesthetized withpentobarbital (50 mg/kg). Blood was harvested by intracardiac puncture,collected in heparinized tubes, and plasma obtained by centrifugation.Mice were perfused with PBS. Brain homogenates were centrifuged at12,000 μg for 10 min and the supernatant acidified by diluting 1:3 with0.1% formic acid (Fluka, Sigma-Aldrich, St. Louis, Mo.). Solid phaseextraction followed by HPLC analysis was used to quantify the amount ofcompound in brain supernatants. Briefly, cartridges (Sep-Pak C18; WatersAssociates, Milford, Mass.) were conditioned with 1 ml of acetonitrile(HPLC grade; EMD Biosciences, San Diego, Calif.) and equilibrated with 1ml of water. A structural analog of MW01-5-188WH was used as an internalstandard. The acidified brain supernatant was added to the cartridgefollowed by a 1 ml wash with 30% acetonitrile. MW01-5-188WH was elutedfrom the cartridge using 80% acetonitrile. The eluate was evaporated todryness, reconstituted in 0.08% formic acid/water in 80% acetonitrile,and analyzed by HPLC using the following gradient in reagent B: 0-60%from 2 to 5 min, isocratic at 65% until 7 min, 65-80% from 7 to 12 min,isocratic at 80% until 15 min, 89-100% from 15 to 18 min, and isocraticat 100% until 23 min. Plasma samples were deproteinized in 0.1 Mperchloric acid and centrifuged at 12,000 μg for 10 min. The supernatantwas neutralized with 1 M NaOH, then extracted with dichloromethane, andthe layers separated at 3000 μg for 5 min. The organic phases from threesuccessive extractions were pooled and then evaporated to dryness underreduced pressure. The dried residue was reconstituted in 50 μl ofreagent B, and 10 μl of the reconstituted material was analyzed by HPLCusing the gradient described above for brain supernatants.

Results: Oral Bioavailability and Brain Uptake of MW01-5-188WH

Integrative chemical biology tools for neurosciences and CNS targeteddrugs must exhibit appropriate bioavailability and brain uptake orpenetration of the blood-brain barrier. Daily oral administration is thepreferred method of administration for longer-term and time-delimited invivo studies using animal models and is the preferred mode in drugdevelopment for a variety of reasons, including better patientcompliance. In this regard, it is critical to demonstratebioavailability and appropriate rates of initial brain uptake for aninhibitor, to fully interpret the outcomes from in vivo studies.Therefore, the rate of MW01-5-188WH concentration change in the bloodafter oral administration (oral bioavailability) and its rate of changein the brain were determined. Using the protocols described above forthe quantitative analysis of MW01-5-188WH extracted from biologicalsamples, the rates of appearance in blood and brain after a low-doseoral administration (2.5 mg/kg) to mice were examined. The appearance ofMW01-5-188WH in the blood (FIG. 35 A) is readily detected within theearliest possible time point, 5 min, with a peak concentration beingreached within 15 min and bulk clearance happening within 120 min afteroral administration. This demonstrates that MW01-5-188WH has good oralbioavailability properties. A similar pattern of time-dependent changein concentration is seen for the brain (FIG. 35B), indicative ofMW01-5-188WH initial brain uptake reflecting that of the blood. However,the MW01-5-188WH peak brain/blood concentration ratio is >3.3,comparable with those of CNS drugs in clinical use. For example, thebrain/blood ratio for minaprine, a 6-phenylaminopyridazine CNS drug, isabout 2 (Caccia et al., 1985 Xenobiotica, 15(12): 1111-9). These resultsdemonstrate that MW01-5-188WH fulfills criteria that typically excludemany compounds that are active in cell culture from being used for invivo investigations and indicate its potential to work in vivo afteroral administration and within the experimental constraints imposed bythe human Aβ ICV infusion model.

MW01-5-188WH Dosing is Selective for CNS Inflammation

The de novo focus on suppression of selected glia activation pathwaysand the excellent brain uptake properties of orally administeredMW01-5-188WH raised the possibility that the compound might exhibitselectivity for CNS proinflammatory cytokine suppression versussuppression of proinflammatory cytokine production by peripheraltissues. To examine this possibility, MW01-5-188WH was administereddaily at a standard therapeutic dose (2.5 mg/kg) by oral gavage for 2weeks, and then mice were challenged with an intraperitoneal injectionof bacterial LPS. Six hours after the LPS challenge, the serum and brainlevels of IL-1β and TNF-α were measured. As anticipated, the LPSchallenge induced an increase in the levels of IL-1β and TNF-α in theserum (FIG. 35C,D) and brain (FIG. 35E,F), compared with control miceinjected with saline. The interesting finding was that treatment withMW01-5-188WH for 2 weeks suppressed the LPS-induced upregulation ofIL-1β and TNF-α production in the brain (FIG. 35E,F) but did notsuppress the serum response (FIG. 35 C,D). The suppression of braincytokine responses by MW01-5-188WH is consistent with its ability tosuppress proinflammatory cytokine production by activated glia and itsoral bioavailability and brain uptake properties shown above.

Example 8 Pharmacokinetics Studies

Plasma Pharmacokinetics and Absolute Bioavailability in Dog and/or Rat

Two groups (3 animals per group; male animals) will be dosed PO and IV.There will be one dose level (2.5 mg/kg), and a crossover design will beused with 1 week washout between dose periods. Plasma drugconcentrations will be measured at not less than eight time points notexceeding 24 hrs post-dose (e.g. 15, 30, 60, 90, 120, 240, and 480minutes and 24 hr after administration of single dose). PK parametersthat will be derived include C_(max), T_(max), t_(1/2), AUC, CI/F, V_(d)and MRT. Dosing formulation: oral gavage/CMC solution.

Mass Balance Study in Dog and/or Rat

A study may be performed utilizing ¹⁴C-labelled minozac (MW01-2-151SRM)to analyze excretion (urine, feces) and plasma distribution.

Dose Range Finding Study in Rat

Phase A of the study will be a single dose MTD (3M/3 F for each doselevel, n=up to MTD or MFD found). Dose levels to be designed based onavailable data if any; doses provided below may be utilized for examplepurposes only. Dosing will be by oral gavage with CMC solution.

Dose level 1: 10 mg/kg; Dose level 2: 100 mg/kg; Dose level 3: 500mg/kg; Dose level 4: 1000 mg/kg; Dose level 5: 3000 mg/kg;Result: An estimated single-dose MTD/MFD (sdMTD)

Phase B of the study may be performed. The phase comprises a 7-day doserange finding study (3M/3 F in each group, n=24). There will be acontrol plus one dose level (a fraction of sdMTD); additional doselevel(s) will be incorporated as required by the outcome of the initial7-day dose range finding study.

Result: An estimated repeat-dose MTD in rats

Dose Range Finding Study in Dog

This study will utilize a single dose MTD (SM crossover study, n=up toMTD or MFD found). Dose levels will be designed based on available data.Examples of doses are provided below. The dosing will be by oral gavagewith a CMC solution preferred; alternatively, filled gelatin capsuleswill be utilized.

Oral dose level 1: 30 mg/kg; Oral dose level 2: 100 mg/kg; Oral doselevel 3: 300 mg/kg

IV dose 1: 100 mg/kg; IV dose 2: 300 mg/kg; Oral dose level 4: 1000mg/kg; Oral dose level 5: 3000 mg/kg.

Each subsequent dosing will be followed by an appropriate washout period(2 days or 5 days after IV exposure). Pharmacokinetics and absolutebioavailability will be determined for dose levels 1 and 2. Plasma drugconcentrations will be measured at eight time points not exceeding 24hrs post-dose (e.g. 15, 30, 60, 120, 240 and 480 minutes afteradministration of single oral doses). PK parameters to be derivedinclude C_(max), T_(max), t_(1/2), AUC, CI/F, V_(d) and MRT.

28-Day Repeat Dose Toxicology Study in Rats

The Main Study will involve 10M/10 F in each treatment group, n=80. Thedosing will be by oral gavage with CMC solution. There will be aControl, Low dose, Mid dose, and High dose. Results: PK (plasma and CSFdrug levels) will be determined at Day 1 and Day 28. Necropsy will bedetermined after the completion of treatment. Mortality, clinicalobservations, body weights, food consumption, clinical pathology,opthalmoscopy, gross pathology, and organ weights will be determined.Histopathology will be determined on control and high dose groups.

A Recovery Study with 5M/5 F in each treatment group, n=20 will beconducted. There will be a Control and High dose. Results: Necropsy willbe determined after 28 days additional follow-up period. Mortality,clinical observations, body weights, food consumption, clinicalpathology, opthalmoscopy, gross pathology, and organ weights will alsobe determined. Histopathology will be determined if required byobservation of treatment effects.

28-Day Repeat Dose Toxicology Study in Dogs

A Main Study utilizing 3M/3 F in each treatment group, n=24 will beconducted. Dosing will be by oral gavage with CMC solution preferred;alternatively, filled gelatin capsules will be used if required. Therewill be a Control, Low dose, Mid dose, and High dose. Results: PK(plasma and CSF drug levels) will be determined at Day 1 and Day 28Necropsy will be determine after the completion of treatment. Mortality,clinical observations, body weights, food consumption, clinicalpathology, gross pathology, and organ weights will be determined.Histopathology will be done on all dose groups

A Recovery Study using 3M/3 F in each treatment group, n=12 will also beconducted. The study will use Control and High dose. Results: Necropsywill be determined after 28 days additional follow-up period. Mortality,clinical observations, body weights, food consumption, clinicalpathology, opthalmoscopy, gross pathology, and organ weights will bedetermined. Histopathology if required will be determined by observationof treatment effects.

Example 9 General Methods

Chemicals were generally purchased from Aldrich (Milwaukee, Wis.) orthrough VWR International and used as received. All solvents were usedas received unless stated otherwise in the text. All organic solutionswere dried with magnesium sulfate before final evaporation. Microwaveirradiation was carried out using the CEM-Discover microwave synthesissystem (Matthews, N.C.).

All intermediates were characterized by MS (ESI) and HPLC and in somecases by ¹H-NMR. Final compounds were characterized by HRMS, HPLC and¹H-NMR, and in some cases, by elemental analysis. NMR spectra wereacquired on a Varian Inova 500 MHz spectrometer at room temperature.Electrospray mass spectra (EI-MS) were collected on a Micromass QuattroII Triple Quadrupole HPLC/MS/MS Mass Spectrometer. High resolution massspectra (HR-MS) were obtained on a VG70-250SE mass spectrometer.

All syntheses were monitored by analytical HPLC. HPLC traces wereobtained on a Rainin Instruments HPLC on commercially available SUPELCOC18 reverse phase column (25×4.6 mm, 5 μm). The mobile phase consistedof 0.1% formic acid in Milli-Q water as reagent A and 0.08% formicacid/Milli-Q water in 80% acetonitrile as reagent B. The flow rate of1.5 ml/min was used in a gradient of 0 to 100% of reagent B over 22minutes. The HPLC traces were tracked by UV absorption at 260 nm.

A separate HPLC system was used to obtain final compound purity. TheHPLC system (Dionex, Sunnyvale, Calif.) consisted of the followingcomponents: a Dionex P680 pump, a Dionex ASI-100 autosampler, aPhenomenex (Torrance, Calif.) Luna C18 column (250×2.0 mm; 5 μm) with aguard column, and a Dionex UVD1700 ultraviolet detector. The mobilephase consisted of 0.1% formic acid in Milli-Q water as reagent A and0.08% formic acid/Milli-Q water in 80% acetonitrile as reagent B. Theflow rate of 0.2 mL/min was used, unless stated otherwise in the text.For determination of compound purity, the gradient consisted of a linearchange from 0 to 100% of reagent B over 30 minutes. UV absorption wasmonitored at four wavelengths (215, 230, 260 and 300 nm) with the 260 nmtrace being reported. Compounds were injected at concentrations100-times greater than the lower detection limit of the instrument (500ng injected).

Elemental analysis was carried out by Quantitative Technologies Inc.(QTI, Whitehouse, N.J.). Melting point data for the dichloro monohydratesalt 26 (234.1-234.7° C.) and of compound 16 (>215° C., decomposes toblack solid) were acquired on a Büchi Melting Point B-540 (Flawil,Switzerland).

Synthesis of R4Analogs (FIG. 37))2-benzyl-6-phenyl-4,5-dihydropyridazin-3(2H)-one (18)

3-benzoylpropionic acid 17 (17.8 g, 0.1 mol), benzylhydrazinedihydrochloride (19.5 g, 0.1 mol) and sodium acetate (74.9 g, 0.55 mol)were suspended in 500 mL ethanol (95%). The white suspension was heatedunder reflux for 29 hours. Ethanol was removed under reduced pressureand the residue was treated with water (300 mL). The pH of the aqueouslayer was adjusted with concentrated solution of sodium carbonate topH=8 and extracted with ethylacetate (1×200 mL). The organic layer waswashed with brine and concentrated to dryness under reduced pressure.The product 18 was obtained as yellow oil in 78% yield and was used inthe following step without further purification. HPLC (t_(r)/purity):23.4 min, 80%.

3,4-dichloro-6-phenylpyridazine (19)

Compound 18 (26 g, 0.079 mol—estimated on 80% purity), phosphorusoxychloride (59 mL, 0.64 mol, 6.5 equiv) and phosphorus pentachloride(133.2 g, 0.64 mol, 6.5 equiv) were heated at 120° C. for 12 hrs. Tocontrol the HCl gas forming during the course of reaction, a NaOHsolution was used to absorb the acid. Most of the phosphoryl chloridewas distilled under reduced pressure, ice water was added to the residueand stirred for 30 min. The yellow crystalline solid which separatedupon cooling was filtered, washed with water (3×100 mL) andrecrystallized from anhydrous ethanol to give desired product 19 asyellow needles in 44% yield. ¹H NMR (CDCl₃): δ 8.06 (dd, ³J=6.5 Hz,⁴J=2.5 Hz, 2H), 7.98 (s, 1H), 7.56 (t, ³J=6.5 Hz, 3H). HPLC (t/purity):23.6 min, >95%.

3-chloro-6-phenylpyridazin-4-ol (20)

A mixture of 19 (158 g, 0.7 mol) and acetic acid (700 mL) was heatedunder reflux for 5 hrs. The reaction mixture was cooled to roomtemperature, the precipitate filtered and the bright yellow filter cakewashed with water (5×500 mL). The filter cake was recrystallized fromethyl acetate (200 mL), filtered and dried over a medium frit sinteredglass funnel in vacuo to give the desired product 20 in 32% yields. HPLC(t_(r)/purity): 15.37 min, >95%. ESI m/z (MeOH): 207.3 (MH⁺).

6-phenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazin-4-ol (21)

Compound 20 (14 g, 0.068 mol) was placed in a reaction tube with1-butanol (30 mL) and 4 equiv of 1-(2-pyrimidyl)piperazine (45 g, 0.27mol, 4 equiv). The flask was capped and heated at 130° C. for 41 h. Thereaction mixture was cooled to ambient temperature, and the 1-butanolremoved under reduced pressure to give a dark oil residue. The oil wastreated with water to give a suspension which is then filtered andwashed with water. The filter cake was dried over a medium frit sinteredglass funnel in vacuo to give the desired product 21 in 97% yields. HPLC(t_(r)/purity): 17.30 min, >99%. ESI m/z (MeOH): 334.38 (MH⁺).

4-chloro-6-phenyl-3-(4-(pyrimidin-2-yl)piperazin-1-yl)pyridazine (6)

Compound 21 (22 g, 0.066 mol) was suspended in phosphorus oxychloride(80 mL). The reaction mixture was heated at 100° C. for 3 h, cooled toroom temperature and poured on crushed ice (2 kg). The aqueous mixturewas neutralized with NaOH solution to give white suspension. Theprecipitate was filtered and dried over a medium frit sintered glassfunnel in vacuo to give the desired product 6 in 91% yields (21 g). ¹HNMR (CDCl₃): δ 8.35 (d, J=4.6 Hz, 2H), 8.01 (d, J=7.5 Hz, 2H), 7.81 (s,1H), 7.50 (t, J=7.0 Hz, 2H), 7.48 (t, J=7.0 Hz, 1H), 6.54 (t, J=4.4 Hz,1H), 4.05 (t, J=4.4 Hz, 4H), 3.65 (t, J=4.4 Hz, 4H). HPLC(t_(r)/purity): 22.4 min, >99%; HRMS calcd for C₁₈H₁₇ClN₆ 352.1198,found 352.1201.

4-Benzyl-6-phenyl-3-(4-pyrimidin-2-yl)piperazin-1-yl)pyridazine (2)

Following the procedure of Zou et al (Tet Lett. 2001 42: 7213-7215),compound 6 (100 mg, 0.28 mmol) was suspended in THF with the 1.37 equivof benzyl boronic acid (42 mg, 0.31 mmol), 0.2 equiv ofPd(dppf)Cl₂CH₂Cl₂ (23 mg, 0.02 mmol), 2.5 equiv of silver oxide (164 mg,0.71 mmol) and 3 equiv of potassium carbonate (117 mg, 0.85 mmol). Themixture was purged with argon and was heated at 120° C. for 16 h in asealed tube. The reaction mixture was then cooled to ambient temperatureand quenched with either 33% hydrogen peroxide or 10% sodium hydroxide.The aqueous layer was extracted with ether (3×30 mL) and the ethereallayers are combined and evaporated under reduced pressure. The crudemixture is run on a silica gel column and eluted with hexanes:ethylacetate (1:1 v/v). The product 2 is obtained as a pale pink solid in 45%yield. ¹H NMR (CDCl₃): δ 8.36 (d, J=4.4 Hz, 2H), 7.94 (d, J=7.1 Hz, 2H),7.46-7.42 (m, 3H), 7.41 (s, 1H), 7.36 (t, J=7.3 Hz, 2H), 7.30 (t, J=7.1Hz, 1H), 7.22 (d, J=7.3 Hz, 2H), 6.55 (t, J=4.4 Hz, 1H), 4.10 (s, 2H),4.01 (s, 4H), 3.44 (s, 4H). HPLC (t_(r)/purity): 30.32 min, >95%; HRMScalcd for C₂₅H₂₄N₆ 408.2057, found 408.2066.

6-Phenyl-4-(pyridin-4-yl)-3-(4-pyrimidin-2-yl)piperazin-1-yl)pyridazine(3)

Compound 6 (700 mg, 2.0 mmol) was placed in a reaction vessel with 3.1equiv potassium carbonate (851 mg, 6.2 mmol), 1.37 equiv (330 mg, 2.7mmol) 4-pyridinylboronic acid and 0.05 equiv Pd(PPh₃)₄ (120 mg, 0.1mmol). DME (10 mL) was added and the mixture was purged with argon. Thereaction mixture was sealed and heated at 110° C. for 20 h. The solutionwas cooled to ambient temperature and filtered through celite. Thefiltrate was concentrated under reduced pressure, dissolved in ethylacetate (30 mL) and washed with 2N HCl (50 mL). The organic layer wasconcentrated under reduced pressure and recrystallized with ethylacetate/petroleum ether mixture to give the product 3 as light yellowneedles in 41% yield. ¹H NMR (CDCl₃): δ 8.79 (d, J=5.5 Hz, 2H), 8.32 (d,J=5.0 Hz, 2H), 8.07 (d, J=7.5 Hz, 2H), 7.68 (d, J=5.5 Hz, 2H), 7.63 (s,1H), 7.51 (t, J=7.0 Hz, 2H), 7.48 (t, J=7.0 Hz, 1H), 6.53 (t, J=4.5 Hz,1H), 3.85 (d, J=4.5 Hz, 4H), 3.39 (t, J=5.0 Hz, 4H). HPLC(t_(r)/purity): 21.61 min, >95%; HRMS calcd for C₂₃H₂₁N₇ 395.1853, found395.1852.

4-Isobutyl-6-phenyl-3-(4-pyrimidin-2-yl)piperazin-1-yl)pyridazine (4)

Following the procedure of Zou et al (supra), compound 6 (200 mg, 0.56mmol) was suspended in THF with the 1.37 equiv of(2-methylpropyl)boronic acid (79 mg, 0.77 mmol), 0.2 equiv ofPd(dppf)Cl₂CH₂Cl₂ (92.5 mg, 0.11 mmol), 2.5 equiv silver oxide (328 mg,1.41 mmol) and 3 equiv of potassium carbonate (234 mg, 1.7 mmol). Themixture was purged with argon and heated at 120° C. for 42 hours in asealed tube. The reaction was cooled to ambient temperature and thereaction was quenched with aqueous solution of sodium hydroxide (10%)and extracted with ether (3×50 ml). The ethereal layers were combined,dried with magnesium sulfate and evaporated under reduced pressureleaving a sticky solid. The crude mixture was purified with columnchromatography and eluted with 40% ethyl acetate in hexanes to give 4 asa white powder in 52.5% yield. ¹H NMR (CDCl₃): δ 8.36 (d, J=4.2 Hz, 2H),8.06 (d, J=7.1 Hz, 2H), 7.60 (s, 1H), 7.51 (t, J=7.0 Hz, 2H), 7.47 (t,J=7.0 Hz, 1H), 6.55 (t, J=4.2 Hz, 1H), 4.03 (s, 4H), 3.42 (s, 4H), 2.62(d, J=6.7 Hz, 2H), 2.18 (sp, J=6.4 Hz, 1H), 0.97 (d, J=6.2 Hz, 6H). HPLC(t_(r)/purity): 29.5 min, >95%; HRMS calcd for C₂₂H₂₆N₆ 374.2213, found374.2208.

4-Methyl-6-phenyl-3-(4-pyrimidin-2-yl)piperazin-1-yl)pyridazine (5)

Following the procedure of Zou et al (supra), compound 6 (250 mg, 0.71mmol) was suspended in THF with the 1.37 equiv of methylboronic acid (59mg, 0.97 mmol), 0.25 equiv of Pd(dppf)Cl₂CH₂Cl₂ (144 mg, 0.18 mmol), 2.5equiv of silver oxide (410 mg, 1.78 mmol) and 3 equiv of potassiumcarbonate (294 mg, 2.1 mmol). The mixture was purged with argon and washeated at 120° C. for 18.5 h in a sealed tube. After cooling to ambienttemperature the reaction was quenched with aqueous sodium hydroxide(10%) and extracted with ether (3×75 ml). The compound was purified bycolumn chromatography and eluted with a mixture of ethyl acetate:hexanes(1:3 v/v). The compound 5 was a white crystallize solid obtained in45.8% yield. ¹H NMR (CDCl₃): δ 8.36 (d, J=4.5 Hz, 2H), 8.05 (d, J=7.5Hz, 2H), 7.61 (s, 1H), 7.50 (t, J=7.1 Hz, 2H), 7.44 (t, J=7.1 Hz, 1H),6.55 (t, J=4.5 Hz, 1H), 4.04 (t, J=4.5 Hz, 4H), 3.46 (t, J=4.5 Hz, 4H),2.45 (s, 3H). HPLC (t_(r)/purity): 24.91 min, >95%; HRMS calcd forC₁₉H₂₀N₆ 332.1744, found 332.1740. Anal. Calcd for C₁₉H₂₀N₆C, 68.65; H,6.06; N, 25.28. Found C, 68.73; H, 5.97; N, 25.22.

Synthesis of R3Analogs (FIG. 38)4-Methyl-6-phenyl-3-(4-pyrazin-2-yl)piperazin-1-yl)pyridazine (7)

Compound 15 (500 mg, 2.4 mmol) was placed in a capped flask andsuspended in 20 mL water. 2.5 equiv (1 g, 6 mmol) of1-(2-pyrazinyl)piperazine and 5 equiv (1.69 mL, 12 mmol) oftriethylamine were added and the flask was capped and heated to 130° C.for 160 h. The reaction was cooled to ambient temperature to give a darkbrown oil at the bottom of the flask. The water was decanted off of theoil, the oil was dissolved in minimal isopropanol and heated to 70° C.Upon cooling, a brown solid formed and was filtered on a sintered glassfunnel and rinsed with hexanes to afford product 7 as a brown powder in28.8% yield. ¹H NMR (CDCl₃): δ 8.25 (bs, 1H), 8.16 (bs, 1H), 8.08 (d,J=7.0 Hz, 2H), 7.93 (bs, 1H), 7.69 (s, 1H), 7.54-7.48 (m, 3H), 3.83 (t,J=5.0 Hz, 4H), 3.57 (bs, 4H), 2.48 (s, 3H). HPLC (t_(r)/purity): HRMScalcd for C₁₉H₂₀N₆, found. Given in Apr. 21, 2006

4-Methyl-6-phenyl-3-(4-pyridin-2-yl)piperazin-1-yl)pyridazine (8)

Compound 15 (190 mg, 0.93 mmol) was placed in a reaction tube with1-butanol and 4 equiv of 1-(pyridin-2-yl)piperazine (605 mg, 3.7 mmol),capped and heated at 140° C. for 48 h. The reaction mixture was cooledto ambient temperature and the 1-butanol removed under reduced pressureto give a dark oil residue. The oil was treated with water to give asuspension which is filtered and washed first with water, then with amixture of ethyl acetate:hexanes (1:6 v/v) to afford the product 8 as abrown yellow powder in 54.5% yield. ¹H NMR (CDCl₃): δ 8.23 (d, J=3.7 Hz,1H), 8.05 (d, J=7.5 Hz, 2H), 7.60 (s, 1H), 7.54 (t, J=6.8 Hz, 1H), 7.49(t, J=7.1 Hz, 2H), 7.44 (t, J=7.3 Hz, 1H), 6.75 (d, J=8.2 Hz, 1H), 6.68(t, J=5.5 Hz, 1H), 3.76 (s, 4H), 3.51 (t, J=4.8 Hz, 4H), 2.43 (s, 3H).HPLC (t_(r)/purity): 15.66 min, >95%; HRMS calcd for C₂₀H₂₁N₅ 331.1791,found 331.1800.

4-Methyl-6-phenyl-3-(4-pyridin-4-yl)piperazin-1-yl)pyridazine (9)

Compound 15 (190 mg, 0.93 mmol) was placed in a reaction tube with1-butanol and 4 equiv of 4-piperazino-pyridazine (605 mg, 3.7 mmol). Theflask was capped and heated at 140° C. for 72 h. The reaction mixturewas cooled to ambient temperature and the 1-butanol removed underreduced pressure to give a dark red oil residue. The oil was treatedwith 20 mL of water, and then extracted with 10 mL of ethyl acetate. Abrown suspension was formed in the organic layer. The precipitate wascollected by filtration and washed with 10 mL of water and then 10 mL ofethyl acetate to afford the product 9 as a brown yellow powder in 34.1%yield. 1H NMR (CDCl₃): δ 8.33 (d, J=4.9 Hz, 2H), 8.06 (d, J=7.1 Hz, 2H),7.64 (s, 1H), 7.52 (t, J=7.6 Hz, 2H), 7.48 (t, J=7.1 Hz, 1H), 6.79 (d,J=5.8 Hz, 2H), 3.58 (s, 4H), 3.56 (s, 4H), 2.45 (s, 3H). HPLC(t_(r)/purity): 14.95 min, >95%; HRMS calcd for C₂₀H₂₁N₅ 331.1791, found331.1799.

3-(4-cyclohexylpiperazin-1-yl)-4-methyl-6-phenylpyridazine (10)

Compound 15 (200 mg, 0.96 mmol) was suspended in 5 mL water with 4 equivcyclohexyl piperazine (651.5 mg, 3.87 mmol) in a 10-mL microwave glassvessel and capped with a septum. Microwave irradiation of 75W was used,the temperature being ramped from room temperature to 175° C. Once 175°C. was reached, the reaction mixture was held at this temperature for 3h. The reaction mixture was allowed to cool to room temperature, thedark brown solution was poured over water to give a suspension, whichwas filtered to afford a beige solid. The solid was washed with 20 mLsaturated sodium bicarbonate to give 10 in 95% yield. ¹H NMR (CDCl₃): δ7.56 (s, 1H), 7.49 (t, J=7.5 Hz, 3H), 7.44 (m, 2H), 3.41 (s, 4H), 2.79(s, 4H), 2.38 (s, 3H), 2.34 (m, 1H), 1.62 (m, 2H), 1.26 (m, 8H). HPLC(t_(r)/purity): PENDING min, >95%; HRMS calcd for C₂₁H₂₈N₄, found GIVENApr. 21, 2006.

4-Methyl-3-(4-methylpiperazin-1-yl)-6-phenylpyridazine (11)

Compound 15 (500 mg, 2.4 mmol) was suspended in 20 mL water in a cappedflask with 4 equiv 1-methyl-piperazine (961 mg, 9.6 mmol). The vesselwas capped and heated at 120° C. for 120 h until complete. The mixturewas cooled to ambient temperature to afford a pale yellow solution witha white solid precipitate. The reaction was filtered, and the aqueousfiltrate washed with ether to remove trace starting materials and thenextracted with ethyl acetate (5×10 mL). The organic washes are combined,dried with magnesium sulfate and the ethyl acetate removed under reducedpressure. The remaining oil was treated with ether and cooled, resultingin the product 11 as yellow needles in 38.7% yield. ¹H NMR (CDCl₃): δ8.02 (d, J=7.0 Hz, 2H), 7.55 (s, 1H), 7.47 (t, J=7.0 Hz, 2H), 7.43 (m,1H), 3.41 (t, J=4.5 Hz, 4H), 2.63 (bs, 4H), 2.38 (s, 3H), 2.36 (s, 3H).HPLC (t_(r)/purity): PENDING 95%; HRMS calcd for C₁₆H₂₀N₄ Given Apr. 21,2006

Synthesis of a Pyrazine analog (FIG. 39)3-methyl-5-phenylpyrazin-2(1H)-one (24)

This compound was prepared following the procedure of Jones (J. Amer.Chem. Soc. 1949, 71, 78-81). Briefly, commercially availablephenylglyoxal 22 (1.02 g, 7.62 mmol) was dissolved in methanol andcooled to −41° C. Commercially available alanine amide 23 (672 mg, 7.62mmol) was dissolved in 25 ml methanol and added to the reaction mixture.A 12.5 N NaOH (0.760 mL, 9.53 mmol) solution was added dropwise whilestirring, maintaining the temperature of the reaction below −10° C. Whenthe addition was complete, the reaction was placed at −5° C. for 2 h.The reaction was then warmed to room temperature and quenched with 12 NHCl solution (0.76 mL), followed by sodium bicarbonate to neutralize thesolution. The methanol was removed under reduced pressure, and theresidue was extracted with chloroform and precipitated with ethylacetate. The compound was isolated as a white powder to give 24 in 18%yield. HPLC (t_(r)/purity): 15.91 min, >97%. ESI m/z (MeOH): 187.35(MH⁺).

2-(4-(3-methyl-5-phenylpyrazin-2-yl)piperazin-1-yl)pyrimidine (25)

This compound was prepared via the pyrazine triflate with1-(2-pyrimidyl)piperazine as the amine following the procedure of Adamset al (Synlett 2004, 11, 2031-2033). Pyridine was used as an anhydrousreagent kept under argon in a sure-seal bottle (Aldrich). The compound24 (100 mg, 0.52 mmol) and DMAP (65.7, 0.52 mmol) were dissolved inpyridine and methylene chloride (0.5: 4 ml v/v), and cooled to 0° C. Thetrifluoromethane sulfonic acid (0.8 mmol, 135.5 μL) was added dropwiseand stirred for 15 min at 0° C. and then 3 h at RT. The triflate wasconfirmed by ESI (363.7 (MH⁺)) and HPLC (t_(R)=25.33 min). The reactionmixture was diluted with dichloromethane and washed one time each with20 ml of water, sodium bicarbonate and brine. The dichloromethane wasremoved under reduced pressure, and the remaining residue was dissolveddirectly in DMSO. 1-(2-pyrimidyl)piperazine (5.3 mmol, 750 μL) was addedand the reaction heated to 60° C. and stirred for 2 h. When complete,the reaction was diluted with ethyl acetate and washed with 1N HCl,after washes with brine and water to remove remaining pyridine. Theorganics were then dried and evaporated in vacuo to give 25 as a yellowsolid (63% yield). HPLC (t_(r)/purity): 24.74 min, >98%. ESI m/z(CH₂Cl₂) 333.29 (MH⁺). ¹H NMR (500 MHz, CDCl₃): δ 8.56 (s, 1H); 8.36 (d,J=4 Hz, 2H); 8.01 (d, J=7.5 Hz, 2H); 7.48-7.40 (m, 3H); 6.54 (bs, 1H);4.03 (bs, 4H), 3.42 (bs, 4H); 2.62 (s, 6H). HRMS calculated GIVEN Apr.21, 2006

Synthesis of 264,6-diphenyl-3-(4-pyrimidin-2-yl)piperazine-1-yl)pyridazine dichloromonohydrate salt (26)

700 mg (1.77 mmol) of 1 was suspended in 10 mL of anhydrous isopropanoland heated to 70° C. 2.5 eq (0.375 mL, 4.4 mmol) of concentrated HCl wasadded at once to the solution. The suspension was stirred at 70° C. for10 min, cooled to ambient temperature and cooled on ice for 1 h. Theprecipitate was collected by filtration and washed once with coldisopropanol (5 mL) to provide the product 26 as bright yellow powder in55% yield. ¹H NMR (DMSO-d₆): δ 8.55 (s, 2H), 8.16 (s, 2H), 7.86 (s, 1H),7.7 (s, 2H), 7.58 (s, 6H), 6.84 (s, 1H), 4.14 (s, 4H), 3.57 (s, 4H).HPLC (t_(r)/purity): PENDING min, >98%. EA calculated for C₂₄H₂₆Cl₂N₆OC59.38, H 5.40, N, 17.31. Found C, 59.38; H, 5.40; N, 17.31.

Production Scheme for Pyrazine Analogs

4,5-dihydro-4-methyl-6-phenylpyridazin-3(2H)-one (13) (Hansen, K B etal. Org. Process Res. Dev., 2005, 9, 634-639, Nelson, D A. US20050137397A1). A 250 ml three-neck round bottom flask fit with atemperature probe and condenser was charged with 7.7 g (40 mmol) of2-methyl-4-oxo-4-phenylbutanoic acid 12 and 20 ml of ethanol (95%). Thesuspension was cooled to below 10° C. and 2.2 ml (42 mmol, 1.05 equiv)of hydrazine monohydrate in 10 ml of ethanol was added dropwise. Afteraddition, the reaction mixture was heated to reflux and stirred for 2 h.The reaction mixture was cooled to ambient temperature and forming whitecrystals were collected by filtration. The solid was then washed with 2NNaHCO₃ (1×30 mL), Milli-Q water (3×60 mL) and dried over a medium fritsintered glass funnel in vacuo to give the desired product 13 in 96.1%yield. ¹H NMR (DMSO-d₆): δ 10.84 (s, 1H), 7.75 (m, 2H), 7.41 (m, 3H),3.12 (m, 1H), 2.60 (m, 1H), 2.50 (m, 1H), 1.13 (d, J=7 Hz, 3H). HPLC(t/purity): PENDING min, >95%; ESI m/z (MeOH) 189.08 (MH⁺)4-methyl-6-phenylpyridazin-3(2H)-one (14) (Csende, F et al. Synthesis,1995, 1240-1242) 7.0 g (35 mmol) of 13 was dissolved in 30 ml ofacetonitrile in a 250 ml single-necked round bottom flask. 11.3 g (84mmol, 2.4 equiv) of anhydrous copper (II) chloride was added to thesolution and the reaction mixture was heated to reflux for 2 hours. Tocontrol the HCl gas that formed during the course of the reaction, aNaOH solution was used to absorb the HCl that escapes from dry tube. Thereaction mixture was cooled to ambient temperature, and placed into anice-water bath. 150 mL of ice-water was added to quench the reaction.The mixture was stirred vigorously for 10 minutes to give a grayprecipitate and blue liquid containing copper (I) chloride. Theprecipitate was then collected by filtration (pH of the filtrate is 0-1)and washed first with 1N HCl (100 mL), then with Milli-Q water (5×100mL). To remove remaining copper by-products, the filter cake was stirredin 1N HCl (150 mL) for 0.5 h and then filtered. The filter cake waswashed with Milli-Q water until the filtrate is at pH 7 (approximately 7washes). The solid was dried over a medium frit sintered glass funnel invacuo to give 14 as a light gray powder in 93.8% yield. ¹H NMR(DMSO-d₆): δ 7.95 (s, 1H), 7.85 (d, J=7.5 Hz, 2H), 7.47 (m, 2H), 7.43(m, 1H), 2.13 (s, 3H). HPLC (t_(r)/purity): 21.48 min, >97%; ESI m/z(MeOH) 187.36 (MH⁺).

3-chloro-4-methyl-6-phenylpyridazine (15)

6.0 g (32 mmol) of 14 was placed in a 250 mL single neck round bottomflask and 30 ml of acetonitrile was added to create a pale yellowslurry. 6.0 ml (64 mmol, 2 equiv) of phosphorus oxychloride was addedand the reaction mixture was heated at reflux for 2.5 h. After thereaction was completed, the mixture was cooled to ambient temperatureand placed in an ice water bath. Ice water (150 mL) was slowly pouredinto the reaction mixture with stirring to decompose the phosphorusoxychloride into HCl and H₃PO₄. The solid was then collected byfiltration and washed with Milli-Q water (3×50 mL). The solid wassuspended in 100 mL of water and 1N NaOH was added until the aqueoussuspension was at pH=8. The mixture was stirred for 5 minutes to removeall trace starting material contaminants. The solid was filtered andwashed with Milli-Q water (3×100 mL). The product was dried over amedium frit sintered glass funnel in vacuo to provide 15 as a light pinkpowder in 96% yield. ¹H NMR (DMSO-d₆): δ 8.29 (s, 1H), 8.10 (m, 2H),7.53 (m, 3H), 2.41 (s, 3H), HPLC (t_(r)/purity): 2888.98 min, >94%.ESIm/z (MeOH) 205.49 (MH⁺).

2-(4-(4-methyl-6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidine (5)

7.5 g (36.6 mmol) of 15 was suspended in 125 mL of Milli-Q water. 60.17g (366.0 mmol, 10 equiv.) of 1-(2-pyrimidyl)piperazine was added and thereaction mixture was heated at reflux with rapid stirring for 60 h. Whencomplete, the reaction mixture was cooled to ambient temperature and twolayers were observed in the flask consisting of an orange aqueous layerand a brown oil that settled to the bottom of the flask. The water wasdecanted off, the oil was dissolved in minimal volume of isopropanol andheated to reflux. After 10 minutes of reflux, the solution was slowlycooled to 0° C. to induce crystallization. Pale yellow crystals werefiltered from isopropanol and rinsed with minimal cold ether to provide5 in 54% yield. ¹H NMR (CDCl₃): δ 8.36 (d, J=4.5 Hz, 2H), 8.05 (d, J=7.5Hz, 2H), 7.61 (s, 1H), 7.50 (t, J=7.1 Hz, 2H), 7.44 (t, J=7.1 Hz, 1H),6.55 (t, J=4.5 Hz, 1H), 4.04 (t, J=4.5 Hz, 4H), 3.46 (t, J=4.5 Hz, 4H),2.45 (s, 3H). HPLC (t_(r)/purity): 24.91 min, >95%; HRMS calcd forC₁₉H₂₀N₆ 332.1744, found 332.1740. Anal. Calcd for C₁₉H₂₀N₆: C, 68.65;H, 6.06; N, 25.28; found C, 68.73; H, 5.97; N, 25.22.

2-(4-(4-methyl-6-phenylpyridazin-3-yl)piperazin-1-yl)pyrimidinedihydrochloride monohydrate salt (16) (Wermuth C G, Stahl P H. SelectedProcedures for the Preparation of Pharmaceutically Acceptable Salts, inStahl PH., Wermuth CG. (Ed.) Handbook of Pharmaceutical Salts,Wiley-VCH, p 249-264). 6.3 g (19.0 mmol) of 5 was suspended in 50 mL ofanhydrous isopropanol and heated to 70° C. 2.5 eq (4.0 mL) ofconcentrated HCl was added at once to the solution. The suspension wasstirred at 70° C. for 10 min, cooled to ambient temperature and cooledon ice 0.5 h. The precipitate is collected by filtration and washed oncewith cold isopropanol (30 mL) to provide the product 16 as a yellowpowder in 93.3% yield. ¹H NMR (DMSO-d₆): δ 8.47 (s, 3H), 8.07 (d, J=4.0Hz, 2H), 7.61 (s, 3H), 6.76 (d, J=2.7 Hz, 2H), 3.99 (s, 4H), 3.60 (s,4H), 2.59 (s, 3H). HPLC (t_(r)/purity): 25.06 min, 99%. HRMS calcd forC₁₉H₂₀N₆ 332.1744, found 332.1744. EA calculated for C₁₉H₂₂Cl₂N₆: C,53.91; H, 5.71; N, 19.85; Cl, 16.75; 0, 3.78. Found C, 53.66; H, 5.52;N, 19.67; Cl, 16.86; 0, 4.12. Copper found to be 2 ppm.

Example 10 Physicochemical Properties Materials/Methods:

The HPLC system (Dionex Corp., Sunnyvale, Calif.) consisted of thefollowing components: a Dionex P680 Pump, a Dionex ASI-100 autosampler,a Phenomenex (Torrance, Calif.) Luna C18 column (250×2.0 mm; 5 μM) witha guard column, and a Dionex UVD170U detector. The mobile phaseconsisted of 0.1% formic acid (Fluka) in Milli-Q water as solvent A and80% acetonitrile (Burdick & Jackson), with 0.08% formic acid in Milli-Qwater as solvent B. Peak quantification was performed based uponabsorption at 254 nm relative to a standard curve obtained by serialdilutions of the compound.

Capillary tubes used in the micro scale aqueous solubility determinationwere purchased from Büchi, Switzerland. The weighting of the compoundswas performed on SartoriusAG (Germany) analytical balance. Milli-Q waterwas obtained using Millipore System (Bedford, Mass.). The orbitalshaker/incubator was purchased from Barnstead International (MelrosePark, Ill.).

Micro Scale Aqueous Solubility Determination

Dry, clean borosilicate capillary tubes were weighed using an analyticalbalance. Between 17-30 mg of 16 was weighed and added to the tubes.Distilled, purified Milli-Q water was added to the tubes to createsolutions with concentrations ranging from 1-2 g/ml. Sample tubes weremixed manually to ensure sufficient wetting and were placed in anincubator set at 37° C. overnight. A sample was collected from eachtube, centrifuged at 10,000 rpm for 10 min, and injected onto areversed-phase HPLC.

Macro Scale Aqueous Solubility Determination

Dry, clean glass Erlenmeyer flasks were weighed using an analyticalbalance. Up to 30 mg of 26 was added to the flasks. Distilled, purifiedwater was added to the flask to create a saturated solution. The flaskswere placed in an orbital shaker/incubator at 37° C., 175 rpm for 72hours. Samples were removed at 24 hour intervals, centrifuged at 10,000rpm for 10 min to remove particulate and injected onto a reversed-phaseHPLC system.

Partition Coefficient Determination

The partition coefficients of 16 and 26 were determined using 1-octanol(Sigma) and water. Between 0.5-1 mg/ml of each compound was dissolved inMilli-Q water and allowed to partition into presaturated octanol. Thesamples were placed horizontally in an orbital shaker/incubator at 37°C. for 1 hour. After 1 h, the samples were centrifuged for 5 min at 1500rpm and the aqueous phase separated. The concentration of compound inboth the aqueous and octanol phases was determined.

Activity Assays

Cell culture assays. Glia cell-based assays of theconcentration-dependent activity of the compounds were done aspreviously described (Hu W, Ralay Ranaivo et al., Current Alzheimer'sResearch 2005, 2:197-205; Mirzoeva S, et al., J Med Chem 2002,45:563-566; Ralay Ranaivo H, et al., J Neurosci 2006, 26:662-670). BV-2mouse microglial cells were cultured for one day in multiwell plates andthen treated in serum-free media for 16 hrs with either control bufferor the standard glial activating stimulus lipopolysaccharide (LPS, fromSalmonella typhimurium; 100 ng/ml) in the presence of diluent ordifferent concentrations of compounds. The accumulation of nitrite, thestable metabolite of nitric oxide (NO), was measured in BV-2 conditionedmedia by the Griess assay as previously described (Hu W, Ralay Ranaivoet al., Current Alzheimer's Research 2005, 2:197-205; Mirzoeva S, etal., J Med Chem 2002, 45:563-566; Mirzoeva S, et al., Brain Res 1999,844:126-134). Levels of IL-1β, TNFα, MCP-1 and IL-1β in cell lysateswere measured by the Mesoscale Discovery system as per themanufacturer's instructions. Cell lysates were analyzed by Western blotsas described (Mirzoeva S, et al., J Med Chem 2002, 45:563-566; RalayRanaivo H, et al., J Neurosci 2006, 26:662-670) to determine the levelsof inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).Results for compounds of the invention are shown in Table 1.

Oral Bioavailability and Brain Uptake

To estimate oral bioavailability (concentration of compound in the bloodas a function of time after oral administration) and to gain insightinto potential brain uptake, compound 5 (2.5 mg/kg) was administered tomice by oral gavage in a 0.5% (w/v) carboxymethylcellulose suspension(Ralay Ranaivo H, et al., J Neurosci 2006, 26:662-670). At 5, 15, 30, 60and 120 min after oral administration, mice were sacrificed, perfusedand their blood and brain were harvested. Brains were homogenized inacetonitrile and then centrifuged at 12000×g for 10 minutes. Next, theplasma and the brain supernatant were acidified by diluting with 0.1%formic acid (Fluka) 1:1 and 1:3, respectively. Solid phase extractionfollowed by HPLC analysis was used to quantify the amount of compound inthe plasma brain supernatants. Briefly, cartridges (Sep-Pak® C18,Waters) were conditioned with 1 ml of acetonitrile (HPLC grade, EMDBiosciences) and equilibrated with 1 ml of water. A structural analog,6-methyl-4-phenyl-3-(4(pyrimidin-2-yl)piperazin-1-yl)pyridazine(MW01-7-057WH), was used as an internal recovery standard. Acidifiedsamples were loaded to the cartridge followed by a 1 ml wash with 10%acetonitrile. Compound 5 was eluted from the cartridge using 80%acetonitrile. The eluate was evaporated to dryness, reconstituted in0.08% formic acid/water in 80% acetonitrile and analyzed by HPLC with0.1% formic acid in water as reagent A and 0.1% formic acid inacetonitrile as reagent B using the following gradient in reagent B: 0%to 50% to 3 min, isocratic at 50% until 6 min, 50% to 70% from 6 to 10min, isocratic at 70% until 13 min, 70% to 80% from 13 to 18 min,isocratic at 80% until 21 min, 80% to 70% from 21 to 23 min, and finallyreturning from 70% to 0% from 23 to 28 min.

In vivo efficacy studies in mice. The study design and treatmentparadigm for intracerebroventricular (ICV) infusion of human oligomericAβ₁₋₄₂ into the mouse were as described previously (Craft J M, et al.,Neurobiol Aging 2004, 25,: 1283-1292) except that compoundadministration was by mouth (Ralay Ranaivo H, et al., J Neurosci 2006,26:662-670). It was previously shown that Aβ-induced neuroinflammationis an early event associated with the onset and progression ofpathophysiology, and can be suppressed by an inhibitor of glialactivation. Female C57Bl/6 mice (Harlan) weighing 20-25 g (3-4 monthsold) were housed in a pathogen free facility under an approximate 12h/12 h dark and light cycle and had access ad libitum to food and water.All animal procedures were approved by the Northwestern Animal Care andUse Committee.

Mice were administered by oral gavage either compound 5 (2.5 mg/kg/day)or solvent control (10% DMSO) in a 0.5% (w/v) carboxymethylcellulosesuspension, once per day treatment began at day 21 after start of Aβ ICVinfusion and continued for 14 days (Ralay Ranaivo H, et al., J Neurosci2006, 26:662-670). Beginning at day 50 after start of Aβ ICV infusion,the Y maze test of spontaneous alternation was used to evaluatehippocampus-dependent spatial learning as described previously (RalayRanaivo H, et al., J Neurosci 2006, 26:662-670). At day 60 after startof Aβ ICV infusion, mice were sacrificed, perfused with a HEPES buffer(10 mM, pH 7.2) containing a protease inhibitor cocktail and brain washarvested and dissected as described previously (Ralay Ranaivo H, etal., J Neurosci 2006, 26:662-670). Levels of IL-1β and TNFα, S100B,synaptophysin, PSD-95, levels in hippocampal supernatants were measuredas previously described (Ralay Ranaivo H, et al., J Neurosci 2006,26:662-670; Craft J M, et al., Neurobiol Aging 2004, 25: 1283-1292;Eldik, L J, 1994).

Immunohistochemical detection of GFAP-positive activated astrocytes andF4/80 positive microglia was performed on 10 μm sections as describedpreviously (Ralay Ranaivo H, et al., J Neurosci 2006, 26:662-670; CraftJ M, et al., Neurobiol Aging 2004, 25: 1283-1292).

Statistical analyses. Experimental and control groups were comparedusing one-way ANOVA with Newman-Keuls post-hoc analysis using astatistical software package (GraphPad Prism version 4.00, GraphPadSoftware, San Diego Calif.). Statistical significance was assumed whenp<0.05.

The present invention is not to be limited in scope by the specificembodiments described herein, since such embodiments are intended as butsingle illustrations of one aspect of the invention and any functionallyequivalent embodiments are within the scope of this invention. Indeed,various modifications of the invention in addition to those shown anddescribed herein will become apparent to those skilled in the art fromthe foregoing description and accompanying drawings. Such modificationsare intended to fall within the scope of the appended claims.

All publications, patents and patent applications referred to herein areincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. All publications, patents and patent applicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the methods etc. which are reported thereinwhich might be used in connection with the invention. Nothing herein isto be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

TABLE 1 Medicinal chemistry refinement

log log IL- R4 R3 MW* S* P* 1β^(⊥) NO^(⊥) 1

394.47 −5.40 3.88 2.5 ± 1.6 >25 2

408.50 −5.33 3.82 5.3 ± 0.6 >25 3

395.46 −4.41 2.40 25.8 ± 7.0  >25 4

374.48 −4.89 3.71 6.1 ± 2.5 22 ± 3  5 CH₃

332.40 −4.08 2.29 8.3 ± 5.8 >25 6 Cl

352.82 −4.64 2.76 9.5 ± 4.0 19 ± 8  7 CH₃

332.40 −1.48 2.01 46.1 ± 23.3 8 CH₃

331.41 −2.11 2.45 7.6 ± 2.9 >25 9 CH₃

331.41 −2.09 2.38 17.7 ± 7.2 >25 10 CH₃

336.47 −2.80 3.88 31.4 ± 4.9  11 CH₃ CH₃ 268.36 −1.22 1.83 48.9 ± 22.2*Calculated using ACD/Solubility DB 9.03. logS is intrinsic solubilityof neutral form of compounds. PSA: 1,2,4-7 = 58.04; 3 = 70.93; 8,9 =45.15; 10,11 = 32.26. ^(⊥)Concentration (μM) required for 50%inhibition⁸. IL-1β = interleukin-1β; NO = nitric oxide.

TABLE 2 Compounds of the formula II Compound Final Code

MWol-2-069A-SRM

MW01-6-127WH

MW01-6-189WH

WH 151SRM

MW01-2-069A- SRM

MW01-1-030A- LKM

MW01-2-127LKM

MW01-2-134LKM

MW01-2-023SRM

MW01-2-141SRM

MW01-2-163MAS

MW01-3-024SRM

MW01-3-027SRM

MW01-7-100WH

MW01-2103LPI

1. A composition effective to provide lower risk of side effects and/ora beneficial pharmacokinetic profile following treatment of a subjectsuffering from a neuroinflammatory disease comprising a therapeuticallyeffective amount of a compound of formula I:

wherein R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹², R¹³, and R¹⁴ are independentlyhydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy,aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl,acyloxy, amino, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl,nitro, cyano, halo, sulfate, sulfenyl, sulfinyl, sulfonyl, sulfonate,sulfoxide, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S, phosphonate,ureido, carboxyl, carbonyl, carbamoyl, or carboxamide; and X isoptionally substituted pyrimidinyl or pyridazinyl, an isomer, apharmaceutically acceptable salt, or derivative thereof.
 2. Acomposition according to claim 1 wherein R¹, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹,R¹², R¹³, and R¹⁴ are independently hydrogen, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy, C₃-C₁₀cycloalkyl, C₄-C₁₀cycloalkenyl, C3-C₁₀cycloalkoxy, C₆-C₁₀aryl,C₆-C₁₀aryloxy, C₆-C₁₀aryl-C₁-C₃alkoxy, C₆-C₁₀aroyl, C₆-C₁₀heteroaryl,C₃-C₁₀heterocyclic, C₁-C₆acyl, C₁-C₆acyloxy, —NH₂, —NHR²⁸, —NR²⁸R²⁹,═NR²⁸, —S(O)₂R²⁸, —SH, —SO₃H, nitro, cyano, halo, haloalkyl, haloalkoxy,hydroxyalkyl, —CO₂H, —CO₂R²⁸, —NHC(O)R²⁸, —C(O)NH₂, —C(O)NHR²⁸,—C(O)NR²⁸R²⁹, —NHS(O)₂R²⁸, wherein R²⁸ and R²⁹ are independentlyselected from C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₁₀cycloalkyl,C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀ aryl C₁-C₃alkyl, C₆-C₁₀heteroaryl and C₃-C₁₀heterocyclic.
 3. A composition according to claim 1comprising a therapeutically effective amount of a compound of theformula II:

wherein R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, andR¹⁷ are independently hydrogen, hydroxyl, alkyl, alkenyl, alkynyl,alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl,cycloalkynyl, cycloalkoxy, aryl, aryloxy, arylalkoxy, aroyl, heteroaryl,heterocyclic, acyl, acyloxy, amino, imino, azido, thiol, thioalkyl,thioalkoxy, thioaryl, nitro, cyano, halo, sulfoxide, sulfenyl, sulfinyl,sulfonyl, sulfonate, sulfate, silyl, silyloxy, silylalkyl, silylthio,═O, ═S, phosphonate, ureido, carboxyl, carbonyl, carbamoyl, orcarboxamide; or an isomer, a pharmaceutically acceptable salt, orderivative thereof.
 4. A composition according to claim 3 wherein R⁴,R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵R¹⁶, and R¹⁷ areindependently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy, C₃-C₁₀ cycloalkyl,C₄-C₁₀cycloalkenyl, C₃-C₁₀cycloalkoxy, C₆-C₁₀aryl, C₆-C₁₀aryloxy,C₆-C₁₀aryl-C₁-C₃alkoxy, C₆-C₁₀aroyl, C₆-C₁₀heteroaryl,C₃-C₁₀heterocyclic, C₁-C₆acyl, C₁-C₆acyloxy, —NH₂, —NHR²⁸, —NR²⁸R²⁹,═NR²⁸, —S(O)₂R²⁸, —SH, —SO₃H, nitro, cyano, halo, haloalkyl, haloalkoxy,hydroxyalkyl, —CO₂H, —CO₂R²⁸, —NHC(O)R²⁸, —C(O)NH₂, —C(O)NHR²⁸,—C(O)NR²⁸R²⁹, —NHS(O)₂R²⁸, wherein R²⁸ and R²⁹ are independentlyselected from C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₁₀cycloalkyl,C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀ aryl C₁-C₃alkyl, C₆-C₁₀heteroaryl and C₃-C₁₀heterocyclic.
 5. A composition according to claim 1wherein R¹ is alkyl, cycloalkyl, or heteroaryl.
 6. A compositionaccording to claim 1 wherein R¹ is:

wherein R¹⁵, R¹⁶ and R¹⁷ are independently hydrogen, hydroxyl, alkyl,alkenyl, alkynyl, alkylene, alkenylene, alkoxy, alkenyloxy, cycloalkyl,cycloalkenyl, cycloalkoxy, cycloalkynyl, aryl, aryloxy, arylalkoxy,aroyl, heteroaryl, heterocyclic, acyl, acyloxy, amino, imino, azido,thiol, thioalkyl, thioalkoxy, thioaryl, nitro, cyano, halo, sulfoxide,sulfate, sulfonyl, sulfenyl, sulfinyl, sulfonate, silyl, silyloxy,silylalkyl, silylthio, ═O, ═S, phosphonate, ureido, carboxyl, carbonyl,carbamoyl, or carboxamide.
 7. A composition according to claim 6,wherein R¹⁵, R¹⁶ and R¹⁷ are independently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy,C₃-C₁₀ cycloalkyl, C₄-C₁₀cycloalkenyl, C₃-C₁₀cycloalkoxy, C₆-C₁₀aryl,C₆-C₁₀aryloxy, C₆-C₁₀aryl-C₁-C₃alkoxy, C₆-C₁₀aroyl, C₆-C₁₀heteroaryl,C₃-C₁₀heterocyclic, C₁-C₆acyl, C₁-C₆acyloxy, —NH₂, —NHR²⁸, —NR²⁸R²⁹,═NR²⁸, —S(O)₂R²⁸—SH, —SO₃H, nitro, cyano, halo, haloalkyl, haloalkoxy,hydroxyalkyl, —CO₂H, —CO₂R²⁸, —NHC(O)R²⁸, —C(O)NH₂, —C(O)NHR²⁸,—C(O)NR²⁸R²⁹, —NHS(O)₂R²⁸, wherein R²⁸ and R²⁹ are independentlyselected from C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl, C₃-C₁₀cycloalkyl,C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀aryl C₁-C₃alkyl, C₆-C₁₀heteroaryland C₃-C₁₀heterocyclic.
 8. A composition effective to provide lower riskof side effects and/or a beneficial pharmacokinetic profile followingtreatment in a subject suffering from a neuroinflammatory diseasecomprising a therapeutically effective amount of a compound of theformula III:

wherein R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, andR¹⁷ hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene, alkenylene,alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, cycloalkynyl, cycloalkoxy,aryl, aryloxy, arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl,acyloxy, amino, imino, azido, thiol, thioalkyl, thioalkoxy, thioaryl,nitro, cyano, halo, sulfoxide, sulfate, sulfonyl, sulfenyl, sulfinyl,sulfonate, silyl, silyloxy, silylalkyl, silylthio, ═O, ═S, phosphonate,ureido, carboxyl, carbonyl, carbamoyl, or carboxamide; with the provisothat R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷cannot all be hydrogen, or an isomer, a pharmaceutically acceptablesalt, or derivative thereof, or an isomer, a pharmaceutically acceptablesalt, or derivative thereof.
 9. A composition according to claim 8wherein R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, andR¹⁷ are independently hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆alkynyl, C₁-C₆alkoxy, C₂-C₆ alkenyloxy, C₃-C₁₀ cycloalkyl,C₄-C₁₀cycloalkenyl, C₃-C₁₀cycloalkoxy, C₆-C₁₀aryl, C₆-C₁₀aryloxy,C₆-C₁₀aryl-C₁-C₃alkoxy, C₆-C₁₀aroyl, C₆-C₁₀heteroaryl,C₃-C₁₀heterocyclic, C₁-C₆acyl, C₁-C₆acyloxy, —NH₂, —NHR²⁸,—NR²⁸R²⁹═NR²⁸, —S(O)₂R²⁸, —SH, —SO₃H, nitro, cyano, halo, haloalkyl,haloalkoxy, hydroxyalkyl, —CO₂H, —CO₂R²⁸, —NHC(O)R²⁸, —C(O)NH₂,—C(O)NHR²⁸, —C(O)NR²⁸R²⁹, —NHS(O)₂R²⁸ wherein R²⁸ and R²⁹ areindependently selected from C₁-C₆alkyl, C₂-C₆alkenyl, C₂-C₆alkynyl,C₃-C₁₀cycloalkyl, C₄-C₁₀cycloalkenyl, C₆-C₁₀aryl, C₆-C₁₀aryl C₁-C₃alkyl,C₆-C₁₀ heteroaryl and C₃-C₁₀heterocyclic.
 10. A composition according toclaim 8 wherein in the compound of the formula III R⁴, R⁵, R⁶, R⁷, R⁸,R⁹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are hydrogen, hydroxyl, alkyl, andone or both of R¹⁰ and R¹¹ are independently substituted orunsubstituted hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, alkylene,alkenylene, alkoxy, alkenyloxy, cycloalkyl, cycloalkenyl, aryl, aryloxy,arylalkoxy, aroyl, heteroaryl, heterocyclic, acyl, acyloxy, sulfonyl,sulfinyl, sulfenyl, amino, imino, azido, thiol, thioalkyl, thioalkoxy,thioaryl, nitro, ureido, cyano, halo, silyl, silyalkyl, silyloxy,silylthio, ═O, ═S, carboxyl, carbonyl, or carbamoyl, or an isomer or apharmaceutically acceptable salt thereof.
 11. A composition according toclaim 8 wherein in the compound of the formula III one of R¹⁰ and R¹¹ isalkyl, in particular C₁-C₆ alkyl and the other of R¹⁰ and R¹¹ ishydrogen.
 12. A composition according to claim 8 wherein in the compoundof the formula III one of R¹⁰ and R¹¹ is aryl, and the other of R¹⁰ andR¹¹ is hydrogen.
 13. A composition according to claim 8 wherein in thecompound of the formula III one of R¹⁰ and R¹¹ is a heteroaryl inparticular an unsaturated 5 to 6 membered heteromonocyclyl groupcontaining 1 to 4 nitrogen atoms, and the other of R¹⁰ and R¹¹ ishydrogen.
 14. A composition according to claim 8 wherein in the compoundof the formula III R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶,and R¹⁷ are hydrogen, and R¹¹ is alkyl, alkenyl, alkynyl, alkylene,alkoxy, aryl, or an unsaturated 5 to 6 membered heteromonocyclyl groupcontaining 1 to 4 nitrogen atoms.
 15. A composition according to claim 8wherein in the compound of the formula III R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are hydrogen and R¹¹ is alkyl orpyridinyl.
 16. A composition according to claim 8 wherein the compoundof the formula III is4-methyl-6-phenyl-3-(4-pyrimidin-2-ylpiperazin-1-yl)pyridazine.
 17. Acomposition according to claim 1 comprising a therapeutically effectiveamount of a compound to selectively reduce or block up-regulation ofIL-1β and S100B, and/or reduce or prevent loss of PSD-95 and/orsynaptophysin.
 18. A composition according to claim 1 comprising atherapeutically effective amount of compound of the formula I, II or IIIto treat a neuroinflammatory disease while reducing inhibitory activityat hERG potassium channel.
 19. A composition according to claim 1comprising a therapeutically effective amount of a compound of theformula I, II or III to treat a neuroinflammatory disease while reducinghERG inhibition.
 20. A composition according to claim 1 wherein thetherapeutically effective amount is effective to selectively reduce orblock up-regulation of IL-1β and S100B, reduce or prevent loss of PSD-95and/or synaptophysin over a dosing period.
 21. A composition accordingto claim 1 comprising a therapeutically effective amount of a compoundof the formula I, II or III suitable for administration to a subject toprovide effective concentrations of the compound in an environment ofuse or an effective dose that results in therapeutic effects in theprevention, treatment, or control of symptoms of a disease disclosedherein.
 22. A composition according to claim 21 wherein the disease is aneuroinflammatory disease.
 23. A composition according to claim 1comprising a dose of compound of formula I, II or III of about 0.1 to100 mg/kg, 0.1 to 50 mg/kg, 0.1 to 25 mg/kg, 0.1 to 20 mg/kg, 0.1 to 15mg/kg, 0.1 to 10 mg/kg, 0.1 to 5 mg/kg, 0.1 to 4 mg/kg, 0.1 to 3 mg/kg,0.1 to 2 mg/kg, or 0.1 to 1 mg/kg.
 24. A method of treating aneuroinflammatory disease in a subject comprising administering acomposition of claim 1 to the subject.
 25. Use of at least one compoundof the formula I, II, or III as defined in claim 1 for the preparationof a medicament for providing lower risks of side effects and/or abeneficial pharmacokinetic profile in treating a neuroinflammatorydisease.
 26. A kit comprising one or more composition of claim 1, acontainer, and instructions for use.